HK1200285B - Composition and method of phytonutrients for metabolic programming effects - Google Patents

Description

Compositions and methods for metabolic programming of plant nutrients

Technical Field

The present disclosure relates to methods of promoting phase II enzyme gene expression in a pediatric subject, the methods comprising administering to the subject a composition comprising an effective amount of a phytonutrient. The present disclosure also relates to milk-based nutritional compositions for pediatric subjects. More specifically, the present disclosure relates to nutritional compositions, in particular infant formulas and growth-stage milk, for pediatric subjects, said products comprising phytonutrients, such as polyphenols, isothiocyanates, carotenoids and mixtures thereof. The composition is capable of promoting phase II enzyme gene expression in the subject. The present disclosure also relates to a nutritional supplement for pregnant or lactating women, the supplement comprising the phytonutrient described above. The supplement is capable of promoting phase II enzyme gene expression in a prenatal infant of the pregnant woman or an infant nursing from the lactating woman.

Background

Phytonutrients, such as polyphenols, carotenoids, and isothiocyanates, are plant-derived bioactive compounds that are associated with various health benefits in adults, including antioxidant activity, improved cardiovascular health, anti-inflammation, anti-aging, and neurological benefits. For example, phytonutrients are believed to provide important health benefits to humans, including protection against oxidative stress, inflammation, and many chronic and degenerative diseases.

For example, polyphenols, such as flavonoids, flavonols, flavones, isoflavones, anthocyanins, and proanthocyanidins, have been shown to have antioxidant and anti-inflammatory activities consistent with promoting vascular health, bone health, and cognitive function. Similarly, the health benefits of carotenoids are known, particularly for ocular health. Additional health benefits of certain carotenoids, such as lutein, zeaxanthin, and lycopene, are also being investigated, including antioxidant activity, cardiovascular protection, and eye and skin health. In addition, isothiocyanates found in cruciferous vegetables are known for their anti-cancer, anti-diabetic and anti-microbial activity.

In addition, phytonutrients are also present to varying degrees in human milk. For example, the carotenoid content of human milk is the subject of many studies, and it is generally concluded that human milk content is directly proportional to the maternal diet and closely related to the carotenoid levels in plasma. Therefore, breast-fed infants are routinely exposed to phytonutrients.

While the underlying mechanisms of potential health benefits remain unclear, phytochemicals are believed to have anti-inflammatory efficacy, act as cell signaling molecules, block the cell cycle, and manipulate phase I and phase II detoxification enzymes. Phase I enzymes include cytochrome enzymes responsible for oxidase activity of mixed functions, while phase II enzymes are often involved in coupled reactions necessary for drug metabolism or further metabolism of the phase I enzyme product. At least 10 families of phase I enzymes have been described in humans. Phase II metabolic enzymes, such as glutathione transferases (GSTs), UDP-glucuronosyltransferases (UGTs), sulfotransferases, N- & O-methyltransferases and nad (p) H: quinone oxidoreductase 1 (NQO1), a potentially harmful substance (xenobiotics) that is capable of being metabolized and ultimately excreted in the adult human body. Phase II coupling reactions generally convert harmful substances into water soluble compounds that can be excreted via urine or bile after phase I activation. There are several types of coupling reactions in vivo, including glucuronidation and sulfation. Prior to the present disclosure, it was not suggested that dietary phytochemicals could regulate the expression of these phase II enzymes in pediatric subjects at different stages of development, such as infants and children, to provide metabolic programming.

Over the past two decades, metabolic programming (imprinting) has gained wide acceptance, but many studies have focused on primary metabolic events leading to late stage obesity and other metabolic disorders. In contrast, the potential effects of early exposure to dietary components of phytochemicals have been little studied.

Despite health benefits of eating fruits and vegetables, dietary levels of phytonutrients in adults and children are often sub-optimal. The infants were not stimulated by most plant bioactive compounds until weaning when non-milk based foods were introduced. However, there is a need in infants to be able to express phase II metabolic enzymes to prevent the accumulation of potential toxins. Therefore, there is a need to optimize the nutrition of pediatric subjects, such as infants and/or children, early in development by including phytonutrients in the diet of infants, children and/or pregnant and lactating women to achieve protection against harmful substances.

Disclosure of Invention

In one embodiment, the present disclosure relates to a method of promoting phase II enzyme gene expression in a pediatric subject comprising administering to the subject a nutritional composition comprising an effective amount of a phytonutrient. In another embodiment, the disclosure relates to a method of promoting phase II enzyme gene expression in an infant nursing from a nursing woman, comprising administering to the nursing woman a composition comprising an effective amount of a phytonutrient, and feeding the infant with breast milk from the nursing woman. In another embodiment, the disclosure relates to a method of promoting phase II enzyme gene expression in a prenatal infant, comprising administering to a female pregnant with the prenatal infant an effective amount of a composition comprising phytonutrients. In one embodiment, the above-described method further promotes and/or regulates phase II enzyme protein expression in said subject.

In one embodiment, the present disclosure relates to a milk-based nutritional composition comprising a fat source, a carbohydrate source, a protein source, and a phytonutrient source, wherein the composition is capable of promoting phase II enzyme gene expression in the subject. In another embodiment, the milk-based nutritional composition is further capable of promoting phase II enzyme protein expression in the subject. The plant nutrient source may comprise a polyphenol, an isothiocyanate, a carotenoid, or a mixture thereof. The nutritional composition may further comprise, among other ingredients, a source of long chain polyunsaturated fatty acids, at least one prebiotic, a source of beta-glucan, at least one probiotic, an amount of choline, a source of iron, or any combination thereof.

In another embodiment, the present disclosure relates to a nutritional supplement for a pregnant or lactating female, the supplement comprising a source of phytonutrients, wherein the supplement is capable of promoting phase II enzyme gene expression in a prenatal infant of the pregnant female or an infant nursing from the lactating female. In one embodiment, the supplement further promotes phase II enzyme protein expression. The plant nutrient source may comprise a polyphenol, an isothiocyanate, a carotenoid, or a mixture thereof.

It is to be understood that both the foregoing general description and the following detailed description present embodiments of the disclosure, and are intended to provide an overview or framework for understanding the nature and character of the disclosure as it is claimed. This description is made for the purpose of illustrating the principles and operation of the claimed subject matter. Other and further features and advantages of the present disclosure will be apparent to those skilled in the art upon reading the following disclosure.

Brief Description of Drawings

FIGS. 1A-C: cell viability in 1 month old (a), 2 years old (B) and adult (C) cell models after phytochemical treatment. FIGS. 1A-C depict cell viability in subjects of different ages after phytochemical treatment. More specifically, fig. 1A depicts cell viability in a 1-month-old cell model compared to control cells in the presence of sulforaphane (° C), catechin (■), and quercetin (tangle-solidup) (fig. 1A), fig. 1B depicts cell viability in a 2-year-old cell model (fig. 1B), and fig. 1C depicts cell viability in an adult cell model (fig. 1C). After sulforaphane treatment, a significant loss of cell viability was observed in the 2-year-old cell model (fig. 1B) compared to control cells. No other significant changes in cell viability were observed in response to catechin (■) or quercetin (a). Results are expressed as a percentage of control and mean ± SEM of at least three independent experiments are shown. Represents significantly reduced cell viability compared to control, assessed by one-way ANOVA with post hoc t-assays, p < 0.05.

FIGS. 2A-C: mRNA expression in response to phytochemical treatment. FIGS. 2A-C show the expression of GST, UGT, and NQO1mRNA in a model of 1 month old (black bars), 2 years old (gray bars), and adult (white bars) fibroblasts in the presence of different concentrations of quercetin (FIG. 2A), catechin (FIG. 2B), and sulforaphane (FIG. 2C). More specifically, fig. 2A shows GST, UGT and NQO1mRNA expression in the presence of different concentrations of quercetin in the fibroblast model at 1 month (black bars), 2 years (grey bars) and adult (white bars). Fig. 2B shows the expression of GST, UGT and NQO1mRNA in the presence of different concentrations of catechin in 1 month (black bars), 2 years (grey bars) and adult (white bars) fibroblast models. FIG. 2C shows the expression of mRNA for GST, UGT, and NQO1 in a1 month (black bars), 2 years (gray bars), and adult (white bars) fibroblast model in the presence of different concentrations of sulforaphane. The adult cell model showed a significant dose-dependent increase in GST and UGT mRNA expression after quercetin treatment. Expression of GST and NQO1mRNA was significantly increased in a1 month old cell model. After catechin treatment, infant cell models showed significant increases in GST and NQO1 mRNA. In addition to the significant increase in GST and NQO1 in the infant cell model, significant increases in GST, UGT and NQO1mRNA were observed in the adult cell line when treated with sulforaphane. Results are mean ± SEM of at least three independent experiments and normalized against control = 1. And &representssignificantly increased expression relative to control as assessed by one-way ANOVA with post hoc t-assays, p <0.05 and p <0.01, respectively.

FIGS. 3A-C: effect of phytochemical treatment on phase II enzyme protein expression. Figure 3A depicts an immunoblot of the effect of 24 hour phytochemical treatment (controls as shown, 5, 10 and 20 μ M) and protein expression of phase II enzymes NQO1 (medium grey bars), UGT (light grey bars) and GST (grey black bars) in a1 month old cell model. FIG. 3B depicts a 24 hour effect of phytochemicals and immunoblot of protein expression of phase II enzymes in a 2 year old cell model. Figure 3C depicts an immunoblot of the effect of 24 hour phytochemical treatment and protein expression of phase II enzymes in an adult cell model. All membranes were stripped (stripeped) and re-probed (re-probe) with anti- β -actin antibody to ensure equal loading. The experiment was repeated at least 3 times and the results were relative proportions to β -actin.

Best mode for carrying out the invention

Reference now will be made in detail to implementations of the disclosure, one or more examples of which are set forth below. Each example is given by way of illustration of the nutritional composition of the present disclosure and is not limiting. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the teachings of the disclosure without departing from the scope or spirit of the disclosure. Features illustrated or described as part of one embodiment, for example, can be used with another embodiment to yield a still further embodiment.

Thus, it is intended that the present disclosure cover such modifications and variations as fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present disclosure are disclosed in or are apparent from the following detailed description. One of ordinary skill in the art will understand that the present discussion is a description of exemplary embodiments only and is not intended as limiting the broader aspects of the present disclosure.

By "nutritional composition" is meant a substance or composition that meets at least a portion of the nutritional needs of a subject. The terms "nutrient", "nutritional formula", "enteral nutrient", "nutritional composition" and "nutritional supplement" are used interchangeably throughout this disclosure and refer to enteral formulas, oral formulas, formulas for infants, formulas for pediatric subjects, formulas for children, long-term milk and/or formulas for adults, such as lactating or pregnant women, in liquid, powder, gel, paste, solid, concentrate, suspension or ready-to-use form.

The term "enteral" means through or within the gastrointestinal or digestive tract. "enteral administration" includes oral feeding, intragastric feeding, transpyloric administration, or any other administration into the digestive tract.

By "pediatric subject" is meant a person less than 13 years of age. In some embodiments, a pediatric subject refers to a human subject that is less than 8 years old. In other embodiments, a pediatric subject refers to a human subject between the ages of 1 and 6 years. In still further embodiments, a pediatric subject refers to a human subject between the ages of 6 and 12.

By "infant" is meant a subject whose age is no greater than about 1 year old, and includes infants from 0 to about 12 months. The term infant includes low birth weight infants, very low birth weight infants and premature infants. By "preterm infant" is meant an infant born before the end of the 37 th week of pregnancy. The term infant also includes prenatal infants, such as infants still in utero.

By "child" is meant a subject from about 12 months to about 13 years of age. In some embodiments, the child is a subject between the ages of 1 and 12 years of age. In other embodiments, the term "child(s)" refers to a subject between about 1 and about 6 years of age, or between about 7 and about 12 years of age. In other embodiments, the term "child(s)" refers to any age range between about 12 months and about 13 years of age.

"child nutritional product" refers to a composition that meets at least a portion of the nutritional needs of a child. Growth-stage milk is an example of a nutritional product for children.

By "infant formula" is meant a composition that meets at least part of the nutritional needs of an infant. In the united states, the content of infant formulas is regulated by federal regulations set forth in chapter 21 (21 c.f.r.) parts 100, 106 and 107 of the united states of federal regulations. These regulations define the levels of macronutrients, vitamins, minerals and other ingredients in an effort to mimic the nutritional and other characteristics of human breast milk.

The term "developmental stage dairy" refers to a broad category of nutritional compositions intended for use as part of a diverse diet to support normal growth and development of children between the ages of about 1 and about 6.

"milk-based" is meant to include components taken or extracted from at least the mammary gland of a mammal. In some embodiments, the milk-based nutritional composition comprises a component derived from milk of a domesticated ungulate, ruminant, or other mammal, or any combination thereof. Additionally, in some embodiments, "milk-based" is meant to encompass bovine casein, whey, lactose, or any combination thereof. Additionally, "milk-based nutritional composition" may refer to any composition comprising any milk-derived or milk-based product known in the art.

By "nutritionally complete" is meant a composition that can be used as the sole source of nutrition that will provide substantially all of the required daily amounts of vitamins, minerals, and/or trace elements, as well as protein, carbohydrate, and lipid. In effect, "nutritionally complete" describes a nutritional composition that provides sufficient amounts of carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required to support normal growth and development of a subject.

Thus, by definition, a "nutritionally complete" nutritional composition for preterm infants will provide qualitatively and quantitatively adequate amounts of carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for the growth of preterm infants.

By definition, a "nutritionally complete" nutritional composition for term infants will provide qualitatively and quantitatively sufficient amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for term infant growth.

By definition, a "nutritionally complete" nutritional composition for children will provide, both qualitatively and quantitatively, sufficient amounts of all of the carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for growth of a child.

The term "essential" as applied to nutrients means that the body is unable to synthesize any nutrients in sufficient amounts for normal growth and maintenance of health, and therefore must be supplemented by a meal. The term "conditionally essential" when applied to nutrients means nutrients that must be supplied by the diet when the body has difficulty obtaining sufficient amounts of precursor compounds for endogenous synthesis.

"nutritional supplement" or "supplement" refers to a composition comprising a nutritionally relevant amount of at least one nutrient. For example, the supplements described herein may provide at least one nutrient to a human subject, such as a lactating or pregnant female.

By "probiotic" is meant a microorganism with low or no pathogenicity that exerts beneficial effects on the health of the host.

By "prebiotic" is meant a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of beneficial intestinal bacteria in the gut, selectively reducing intestinal pathogens, or beneficially affecting the intestinal short chain fatty acid profile that improves host health.

"phytonutrients" means chemical compounds that occur naturally in plants. The term "phytonutrients" encompasses several broad classes of compounds produced by plants, such as, for example, polyphenolic compounds, e.g., flavonoids, flavonols, flavones, isoflavones, flavan-3-ols, isoflavonoids, anthocyanins, proanthocyanidins, catechins, and epicatechins. Phytonutrients also encompass carotenoids, phytosterols, thiols, isothiocyanates, and other plant derived compounds.

"beta-glucan" means all beta-glucans, including beta-1,3-glucan and beta-1,3, 1, 6-glucan, each as a specific type of beta-glucan. In addition, the beta-1,3-glucan and the 1, 6-glucan are beta-1, 3-glucan. Thus, "beta-1, 3-glucan" includes beta-1,3, 1, 6-glucan.

All percentages, parts and ratios used herein are by weight of the total composition, unless otherwise specified.

The nutritional compositions of the present disclosure may be free or substantially free of any of the optional or selected ingredients described herein. As used herein, and unless otherwise specified, the term "substantially free" means that the selected composition may contain less than a functional amount of the optional ingredient, typically less than 0.1% by weight, and also including 0% by weight of such optional or selected ingredient.

All references to singular features or limitations of the present disclosure shall include the corresponding plural features or limitations, and vice versa, unless the content of the reference indicates otherwise or clearly indicates the contrary.

All combinations of methods or process steps as used herein can be performed in any order, unless the content of the referenced combination indicates otherwise or clearly contradicted by context.

The methods and compositions of the present disclosure, including components thereof, can include, consist of, or consist essentially of the essential elements and limitations of the embodiments as described herein, as well as any other or optional ingredients, components, or limitations described herein or otherwise useful in nutritional compositions.

As used herein, the term "about" should be understood to mean two numbers recited in any range. Any reference to a range should be considered to provide support for any subset of the ranges.

The present disclosure provides nutritional compositions comprising a plant nutrient source. The nutritional composition may further comprise a protein source, a carbohydrate source, and/or a fat or lipid source. More specifically, the present disclosure provides a milk-based nutritional composition comprising a fat source, a carbohydrate source, a protein source, and a plant nutrient source.

The nutritional compositions of the present disclosure include at least one plant nutrient. The plant nutrient source may be derived from a fruit or vegetable, or in certain embodiments, the plant nutrient source may be a chemical source or synthetically prepared. In some embodiments, the plant nutrient source can comprise a polyphenol, a carotenoid, an isothiocyanate, or a mixture thereof.

For the purposes of this disclosure, phytonutrients may be added to the nutritional composition in their natural, purified, encapsulated, and/or chemically or enzymatically modified forms to deliver desirable organoleptic and stabilizing properties. In the case of encapsulation, it is desirable that the encapsulated phytonutrients resist water dissolution but are released upon reaching the small intestine. This can be achieved by applying an enteric coating, such as cross-linked alginate or the like. Additionally, the nutritional composition may comprise metabolites of plant nutrients or their parent compounds.

Polyphenols suitable for use in the nutritional compositions described herein include, but are not limited to, anthocyanins, proanthocyanidins, anthocyanidins, flavanols, flavonols, flavan-3-ols, flavones, flavanones, and isoflavonoids. For example, the polyphenols include epicatechin, catechin, resveratrol, quercetin, curcumin, or any mixture thereof, as well as any possible combination of phytonutrients in purified or natural form.

In some embodiments, the anthocyanin can be, but is not limited to, a glucoside of aurantiin (aurantidin), anthocyanins, delphinidin, erucic acid pigment (europinidin), luteolin, pelargonidin, malvidin (malvidin), peonidin, morning glory pigment, and rosachrome (rosinidin). These and other anthocyanins suitable for use in the nutritional compositions are found in a variety of plant sources. The anthocyanins can be derived from a single plant source or a combination of plant sources. Non-limiting examples of plants rich in anthocyanins suitable for use in the compositions of the invention include: berries (acai berry, grape, raspberry, blueberry, blackcurrant, chokeberry, blackberry, raspberry, cherry, red currant, cranberry, red berry, cloudberry, blueberry, chokeberry), purple corn, purple sweet potato, purple carrot, sweet potato, red cabbage, eggplant.

Proanthocyanidins suitable for use in the nutritional compositions described herein include, but are not limited to, polymers of flavan-3-ols (e.g., catechins, epicatechins) having a degree of polymerization in the range of 2 to 11. Such compounds may be derived from a single plant source or a combination of plant sources. Non-limiting examples of plant sources rich in proanthocyanidins suitable for the present composition include: grape, grape skin, grape seed, green tea, black tea, apple, pine bark, cinnamon, cocoa, raspberry, cranberry, blackcurrant, chokeberry.

Non-limiting examples of flavanols suitable for use in the nutritional compositions of the present invention include catechin, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, quercetin, myricetin, kaempferol, or any mixture thereof. Plants rich in suitable flavan-3-ols and flavonols include, but are not limited to, apple, grape seed, grape skin, tea (green or black), pine bark, cinnamon, cocoa, raspberry, cranberry, black currant, chokeberry, orange, lime, and lemon.

If the nutritional composition is formulated for administration to a pediatric subject at least 1 year of age, the amount of catechins may range from about 1000 to about 2000 nmol/L. In some embodiments, the nutritional composition is formulated to deliver flavan-3-ols (e.g., catechols) in an amount that may range from about 0.1 to about 170 mg/day. In other embodiments, the nutritional composition is formulated to deliver flavan-3-ols in an amount that may range from about 0.01 to about 150 mg/day. And in certain embodiments, the nutritional composition comprises between about 0.01 and about 338 mg flavan-3-ols per liter. The amount of flavonol (e.g., quercetin) can range from about 50 to about 400 nmol/L. In certain embodiments, the nutritional composition comprises between about 0.01 and about 211mg of flavonol per liter. And in some embodiments, the nutritional composition is formulated to deliver an amount of flavonols (e.g., quercetin) that may range from about 0.1 to about 150 mg/day. If the nutritional composition is related to or formulated for administration to an infant from about 0 to about 12 months of age, the amount of catechins may range from about 500 to about 1300 nmol/L. In some embodiments, the nutritional composition may be formulated to deliver between about 0.01 and about 50 mg of flavan-3-ols, such as catechins, per day. The amount of quercetin can range from about 50 to about 200 nmol/L. In some embodiments, the nutritional composition may be formulated to deliver between about 0.01 and about 40 mg of flavonol, such as quercetin, per day.

In some embodiments, the nutritional compositions of the present disclosure comprise flavanones and/or flavones. Non-limiting examples of suitable flavanones include butein, eriodictyol, hesperetin, hesperidin, homoeriodictyol (horeriodicityl), isosakuranetin, naringenin, naringin, pinocembrin, poncirin, sakuranetin. Non-limiting examples of suitable flavones include apigenin (apigenin) and luteolin (luteolin). Plant sources rich in flavanones and/or flavones include, but are not limited to, orange, citrus, grapefruit, lemon, lime, celery, parsley, and bell pepper. In addition, the nutritional composition may further comprise a flavonoid. The flavonoids from plant or seaweed extracts can be used in the form of monomers, dimers and/or multimers.

The nutritional composition may further comprise an isoflavonoid. Examples of isoflavonoids include, but are not limited to, genistein (genistin), daidzein (daidzin), biochanin A, formononetin, coumestrol (coumestrol), irilone, olopol (orobol), pseudoindoxanthin, anagyroidisoflavanone A and B, calycosin, glycitein, irigenin (irigenin), 5-O-methyl genistein, pratensein, prunetin, psi-tectorigenin, swiss flavone (retusin), tectorigenin, iriridine, formononetin, puerarin, tectoridine, derrubine, luteolin, wyone (wihteone), cattail isoflavone (alpiniusioflavone), barbiger, di-O-methyl tail isoflavone, 4' -methyl cattail isoflavone. Plant sources rich in isoflavonoids include, but are not limited to, psoralea, pueraria, lupin, fava bean, chickpea, alfalfa, and peanuts. In certain embodiments, the nutritional composition may be free or substantially free of soy isoflavonoids. In some embodiments, the nutritional composition may be free or substantially free of soy phytonutrients, such as soy isoflavonoids.

The nutritional composition may comprise other kinds of dietary phytonutrients, such as glucosinolates or isothiocyanates. Representative isothiocyanates include, but are not limited to, sulforaphane and phenethylisothiocyanate. Isothiocyanate rich plant sources include cruciferous vegetables such as brussels sprouts, cabbage, cauliflower, pakchoi, kale, cabbage mustard, turnip, kohlrabi, mustard, radish, rocket, cress, and mixtures thereof.

In certain embodiments, the nutritional composition comprises a carotenoid, such as lutein, zeaxanthin, astaxanthin, lycopene, beta-carotene, alpha-carotene, gamma-carotene, and/or beta-cryptoxanthin. Carotenoid rich plant sources include, but are not limited to, kiwi, grape, citrus, tomato, watermelon, papaya and other red fruits or dark green vegetables such as kale, spinach, radish sprouts, unripe cabbage leaves, lettuce, broccoli, zucchini, pea and brussels sprouts, spinach, carrot and other red, orange or yellow fruits and vegetables.

In some embodiments, the nutritional composition is a fortified milk-based nutritional composition, such as an infant formula or a growth-stage milk, comprising at least one phytonutrient. The nutritional composition may be capable of metabolic programming of phase II enzymes in a pediatric subject. For example, the nutritional composition may be capable of promoting phase II enzyme gene expression and/or phase II enzyme protein expression in a pediatric subject. In some embodiments, the nutritional composition may be capable of modulating phase II enzyme protein expression in a pediatric subject.

The disclosed nutritional compositions may be provided in any form known in the art, such as a powder, gel, suspension, paste, solid, liquid concentrate, reconstitutable powdered formula, or ready-to-use product. In certain embodiments, the nutritional composition may comprise a nutritional supplement, a pediatric nutritional product, an infant formula, a human milk fortifier, a growth-stage milk, or any other nutritional composition designed for pediatric subjects. The nutritional compositions of the present disclosure include, for example, orally ingestible health-promoting substances, including, for example, foods, beverages, tablets, capsules, and powders. In addition, the nutritional compositions of the present disclosure can be standardized to specific caloric content, which can be provided as a ready-to-use product, or in a concentrated form. In some embodiments, the nutritional composition is in the form of a powder having a particle size in the range of 5 μm to 1500 μm, more preferably in the range of 10 μm to 1000 μm, and even more preferably in the range of 50 μm to 300 μm.

In some embodiments, the present disclosure provides fortified milk-based growth-stage milk designed for children aged 1-3 years and/or 4-6 years, wherein the growth-stage milk supports growth and development and life-long health. In some embodiments, the present disclosure provides infant formulas suitable for infants with an age in the range of from 0 to 12 months, or from 0 to 3 months, 0 to 6 months, or 6 to 12 months.

Suitable fat or lipid sources for the nutritional compositions of the present disclosure may be any known or used in the art, including, but not limited to, animal sources, e.g., milk fat, butter oil, milk fat, egg yolk lipids; marine sources, such as fish oils, marine oils, single cell oils; vegetable and vegetable oils, such as corn oil, canola oil, sunflower oil, soybean oil, palm oil essential oil, coconut oil, high oleic sunflower oil, evening primrose oil, rapeseed oil, olive oil, linseed (flaxseed) oil, cottonseed oil, high oleic safflower oil, palm stearin, palm kernel oil, wheat germ oil; emulsions and esters of medium chain triglyceride oils and fatty acids; and any combination thereof.

The carbohydrate source can be any used in the art, for example, lactose, glucose, fructose, corn syrup solids, maltodextrin, sucrose, starch, rice syrup solids, and the like. The amount of carbohydrate in the nutritional composition may generally vary between about 5 g and about 25 g/100 kcal.

The nutritional compositions of the present disclosure may also include a protein source. The protein source may be any used in the art, for example, skim milk, whey protein, casein, soy protein, hydrolyzed protein, amino acids, and the like. Sources of milk protein that may be used in the practice of the present disclosure include, but are not limited to, milk protein powder, milk protein concentrate, milk protein isolate, skim milk solids, skim milk powder, whey protein isolate, whey protein concentrate, sweet whey, acid whey, casein, acid casein, caseinates (e.g., sodium caseinate, sodium calcium caseinate, calcium caseinate), and any combination thereof.

In one embodiment, the protein in the nutritional composition is provided as an intact protein. In other embodiments, the protein is provided as a combination of both intact and partially hydrolyzed proteins. In certain other embodiments, the protein is more completely hydrolyzed. In still further embodiments, the protein source comprises amino acids. In yet another embodiment, the protein source may be supplemented with a glutamine-containing peptide.

In a particular embodiment of the nutritional composition, the protein-derived whey: the casein ratio is similar to that found in human breast milk. In one embodiment, the protein source comprises from about 40% to about 80% whey protein and from about 20% to about 60% casein.

In some embodiments, the nutritional composition comprises between about 1 g and about 7 g of protein source per 100 kcal.

In one embodiment, the nutritional composition may comprise one or more probiotics. In this embodiment, any probiotic known in the art is acceptable. In a particular embodiment, the probiotic may be selected from any of the genera lactobacillus (lactobacillus: (a), (b), (c), and (d)Lactobacillus) Lactobacillus rhamnosus GG (GG)Lactobacillus rhamnosusGG, ATCC No. 53103), Bifidobacterium (II)Bifidobacterium) Species Bifidobacterium longum: (Bifidobacterium longum) And Bifidobacterium animalis subsp lactis BB-12 (Bifidobacterium animalis subsp. lactisBB-12, DSM No. 10140), or any combination thereof.

If included in the composition, the probiotic may be present in an amount of from about 1 x 10 per kg body weight per day⁴To about 1 x 10¹⁰Individual colony forming units (cfu) change. In another embodiment, the probiotic may be present in an amount of from about 10 per kg body weight per day⁶To about 10¹⁰The cfu varies. In yet another embodiment, the amount of probiotic may be from about 10 per day⁷To about 10⁹The cfu varies. In yet another embodiment, the probiotic may be in an amount of at least about 10 per day⁶cfu。

In one embodiment, the probiotic may be viable or non-viable. As used herein, the term "viable" refers to living microorganisms. The term "non-viable" or "non-viable probiotic" means a non-living probiotic microorganism, a cellular component thereof and/or a metabolite thereof. Such non-viable probiotics may be inactivated by heat or otherwise, but they retain the ability to beneficially affect the health of the host. The probiotics useful in the present disclosure may be naturally occurring, synthetic or developed by genetic manipulation of organisms, whether such new sources are now known or later developed.

In certain embodiments, the nutritional composition may further comprise one or more prebiotics. Such prebiotics may be naturally occurring, synthetic or developed by genetic manipulation of organisms and/or plants, whether such new sources are now known or later developed. Prebiotics useful in the present disclosure may include oligosaccharides, polysaccharides, and other prebiotics containing fructose, xylose, soy, galactose, glucose, and mannose.

More specifically, prebiotics useful in the present disclosure may include polydextrose, polydextrose powder, lactulose, lactosucrose (lactosucrose), raffinose, oligoglucose, inulin, fructooligosaccharide, isomaltooligosaccharide, soy oligosaccharide, lactosucrose, xylooligosaccharide, chitooligosaccharide, oligomannose, arabinooligosaccharide, sialyloligosaccharide, fucooligosaccharide, galactooligosaccharide, and gentiooligosaccharide.

In one embodiment, the total amount of prebiotics present in the nutritional composition may be from about 1.0 g/L to about 10.0 g/L of the composition. At least 20% of the prebiotics may comprise galactooligosaccharides, polydextrose, or a mixture thereof. In one embodiment, the amount of each galactooligosaccharide and/or polydextrose in the nutritional composition may be in a range from about 1.0 g/L to about 4.0 g/L.

The nutritional compositions of the present disclosure may include a source of long chain polyunsaturated fatty acids (LCPUFAs) including docosahexaenoic acid. Other suitable LCPUFAs include, but are not limited to, alpha-linoleic acid, gamma-linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), and arachidonic acid (ARA).

In one embodiment, in particular if the nutritional composition is an infant formula, the nutritional composition is supplemented with both DHA and ARA. In this embodiment, the weight ratio of ARA to DHA may be between about 1:3 and about 9: 1. In a specific embodiment, the ratio of ARA to DHA is from about 1:2 to about 4: 1.

The nutritional composition may be supplemented with DHA and/or ARA containing lipids using standard techniques known in the art. For example, DHA and ARA may be added to the composition by replacing an equivalent amount of oil normally present in the composition, such as high oleic sunflower oil. As another example, lipids containing DHA and ARA may be added to the composition by replacing the remaining total fat blend in equal amounts that are normally present in compositions not containing DHA and ARA.

If sources of DHA and/or ARA are included, the source may be any source known in the art, such as marine oil, fishOil, single cell oil, egg yolk lipids and brain lipids. In some embodiments, the DHA and ARA are derived from single cell Martek oil, DHASCO^®And ARASCO^®Or a variant thereof. DHA and ARA can be in natural form, provided that the remaining LCPUFA source does not produce any substantial deleterious effects on the subject. Alternatively, DHA and ARA may be used in refined form.

In one embodiment, the source of DHA and ARA is single cell oil, as taught in U.S. Pat. nos. 5,374,657, 5,550,156, and 5,397,591, the disclosures of which are incorporated herein by reference in their entirety. However, the present disclosure is not limited to only such grease.

The nutritional composition may further comprise a source of beta-glucan. Glucans are polysaccharides, in particular polymers of glucose that occur naturally and are found in the cell walls of bacteria, yeasts, fungi and plants. Beta-glucans (beta-glucans) are themselves a diverse subset of glucose polymers, composed of chains of glucose monomers linked together by beta-type glycosidic bonds to form complex carbohydrates.

Beta-1,3-glucan is a carbohydrate polymer purified from, for example, yeast, mushrooms, bacteria, algae and cereals (Stone BA, Clarke AE. Chemistry and Biology of (1-3) -Beta-glucans, London: Portland Press Ltd; 1993.). The chemical structure of beta-1,3-glucan depends on the source of the beta-1, 3-glucan. In addition, various physicochemical parameters, such as solubility, primary Structure, molecular weight and branching, contribute to the biological activity of β -1,3-glucan (Yadomae T., Structure and biological activities of fungal beta-1,3-glucans. Yakugaku Zasshi. 2000;120: 413-431.).

Beta-1,3-glucan is a naturally occurring polysaccharide with or without the beta-1, 6-glucan side chains found in the cell walls of a variety of plants, yeasts, fungi, and bacteria. Beta-1, 3;1, 6-glucans are those which comprise glucose units with a (1,3) linkage, with a side chain attached at the (1,6) position. Beta-1,3, 1, 6-glucans are a heterogeneous group of glucose polymers that possess a structural commonality comprising a backbone of linear glucose units linked by beta-1,3 bonds, and beta-1, 6 linked glucose branches extending from the backbone. While this is the basic structure of the presently described class of β -glucans, there may be some variations. For example, certain yeast β -glucans have additional regions of β (1,3) branches extending from the β (1,6) branches, which further increases the complexity of their respective structures.

Derived from baker's yeast (Saccharomyces cerevisiae: (A)Saccharomyces cerevisiae) β -glucans consisting of a chain of molecules linking D-glucose at the 1 and 3 positions, with glucose side chains linked at the 1 and 6 positions yeast-derived β -glucans are insoluble, fibrous complex sugars, having the basic structure of a linear chain of glucose units with a β -1,3 backbone interspersed with β -1,6 side chains of typically 6-8 glucose units in length more particularly the β -glucan derived from baker's yeast is poly- (1,6) - β -D-glucopyranosyl- (1,3) - β -D-glucopyranose.

Furthermore, beta-glucan is well tolerated and does not produce or cause excessive gas, abdominal distension, bloating, or diarrhea in pediatric subjects. The addition of beta-glucan to a nutritional composition for a pediatric subject, such as infant formula, growing-period milk, or another children's nutritional product, will improve the subject's immune response by enhancing resistance to invading pathogens, and thus maintain or improve overall health.

In one embodiment, the nutritional composition of the present disclosure comprises choline. Choline is a nutrient that is critical to the normal function of cells. It is a precursor of membrane phospholipids and accelerates the synthesis and release of the neurotransmitter acetylcholine, which is involved in memory storage. Additionally, while not wishing to be bound by this or any other theory, it is believed that dietary choline and docosahexaenoic acid (DHA) act synergistically to promote phosphatidylcholine biosynthesis and thus help promote synaptogenesis in human subjects. Furthermore, choline and DHA may exhibit a synergistic effect promoting dendritic spine formation, which is important in maintaining established synaptic connections. In some embodiments, the nutritional compositions of the present disclosure comprise from about 40 mg choline per serving to about 100 mg per 8 oz serving.

In one embodiment, the nutritional composition comprises a source of iron. In one embodiment, the iron source is ferric pyrophosphate, ferric orthophosphate, ferrous fumarate, or mixtures thereof, and in some embodiments, the iron source may be encapsulated.

One or more vitamins and/or minerals may also be added to the nutritional composition in an amount sufficient to supply the subject's daily nutritional needs. One of ordinary skill in the art will appreciate that the vitamin and mineral requirements will vary based on, for example, the age of the child. For example, an infant may have vitamin and mineral requirements that are different from children between 1 and 13 years of age. Thus, the embodiments are not intended to limit the nutritional composition to a particular age group, but rather to provide a range of acceptable vitamin and mineral components.

In embodiments where a nutritional composition for children is provided, the composition may optionally include, but is not limited to, one or more of the following vitamins or derivatives thereof: vitamin B₁(thiamine, thiamine pyrophosphate, TPP, thiamine triphosphate, TTP, thiamine hydrochloride, thiamine nitrate), vitamin B₂(riboflavin, flavin mononucleotide, FMN, flavin adenine dinucleotide, FAD, lactoflavin, ovalbumin), vitamin B₃(nicotinic acid, nicotinamide adenine dinucleotide, NAD, nicotinic acid mononucleotide, NicMN, pyridine-3-carboxylic acid), vitamin B₃Precursor tryptophan, vitamin B₆(pyridoxine, pyridoxal, pyridoxamine, pyridoxine hydrochloride), pantothenic acid (pantothenate), panthenol, folic acid (folate), pteroylglutamic acid), vitamin B₁₂(cobalamin, mecobalamin, desoxyadenosylcobalamin, cyanocobalamin, hydroxycobalamin, adenosylcobalamin), biotin, vitamin C (ascorbic acid), vitamin A (retinol, retinyl acetate, retinyl palmitate, retinyl esters with other long chain fatty acids, retinolAldehydes, retinoic acid, retinol esters), vitamin D (calciferol, cholecalciferol, vitamin D₃1, 25-dihydroxyvitamin D), vitamin E (α -tocopherol, α -tocopheryl acetate, α -tocopheryl succinate, α -tocopheryl nicotinate, α -tocopherol), vitamin K (vitamin K)₁Phylloquinone, naphthoquinone, vitamin K₂Menaquinone-7, vitamin K₃Menaquinone-4, menaquinone-8H, menaquinone-9H, menaquinone-10, menaquinone-11, menaquinone-12, menaquinone-13), choline, inositol, β -carotene, and any combination thereof.

In embodiments that provide a children's nutritional product, such as a growing-period milk, the composition may optionally include, but is not limited to, one or more of the following minerals or derivatives thereof: boron, calcium acetate, calcium gluconate, calcium chloride, calcium lactate, calcium phosphate, calcium sulfate, chloride, chromium chloride, chromium picolinate (chromium picolinate), copper sulfate, copper gluconate, copper sulfate, fluoride, iron, carbonyl iron, ferric iron, ferrous fumarate, ferric orthophosphate, iron mill, polyferose, iodide, iodine, magnesium carbonate, magnesium hydroxide, magnesium oxide, magnesium stearate, magnesium sulfate, manganese, molybdenum, phosphorus, potassium phosphate, potassium iodide, potassium chloride, potassium acetate, selenium, sulfur, sodium, docusate sodium, sodium chloride, sodium selenate, sodium molybdate, zinc oxide, zinc sulfate, and mixtures thereof. Non-limiting exemplary derivatives of mineral compounds include salts, basic salts, esters, and chelates of any mineral compound.

The minerals may be added to the growing-period dairy product or other children's nutritional composition in the form of salts, such as calcium phosphate, calcium glycerophosphate, sodium citrate, potassium chloride, potassium phosphate, magnesium phosphate, ferrous sulfate, zinc sulfate, copper sulfate, manganese sulfate, and sodium selenite. Additional vitamins and minerals may be added as is known in the art.

In one embodiment, the children's nutritional composition may include between about 10 and about 50% of the highest dietary suggested amount in any given country, or between about 10 and about 50% of the average dietary suggested amount in a group of countries, of vitamin A, C, and E, zinc, iron, iodine, selenium and choline per serving. In another embodiment, the children's nutritional composition may provide from about 10-30% of the highest dietary recommendation amount for any given country, or from about 10-30% of the average dietary recommendation amount for a group of countries, of B vitamins per serving. In yet another embodiment, the levels of vitamin D, calcium, magnesium, phosphorus and potassium in the children's nutritional product may correspond to average levels found in milk. In other embodiments, the other nutrients present in each serving of the children's nutritional composition may be about 20% of the highest dietary recommendation amount for any given country, or about 20% of the average dietary recommendation amount for a group of countries.

The children's nutritional compositions of the present disclosure may optionally include one or more of the following flavors, including, but not limited to, a flavor extract, a volatile oil, a cocoa or chocolate flavor, a peanut butter flavor, cookie crumbs, vanilla or any commercially available flavor. Examples of useful flavoring agents include, but are not limited to, pure anise extract, artificial banana extract, artificial cherry extract, chocolate extract, pure lemon extract, pure orange extract, pure peppermint extract, honey, artificial pineapple extract, artificial rum extract, artificial strawberry extract, or vanilla extract; or volatile oil, such as Melissa oil (balmol), laurel oil, bergamot oil, cedar wood oil, cherry oil, cinnamon oil, clove oil, or peppermint oil; peanut butter, chocolate flavoring, vanilla cookie crumb, cream candy, toffee, and mixtures thereof. The amount of flavoring agent may vary greatly depending on the flavoring agent used. The type and amount of flavoring agent can be selected as is known in the art.

The nutritional compositions of the present disclosure may optionally include one or more emulsifiers added for final product stability. Examples of suitable emulsifiers include, but are not limited to, lecithin (e.g., from egg or soy), alpha-lactalbumin and/or mono-and di-glycerides, and mixtures thereof. Other emulsifiers will be apparent to those skilled in the art and the selection of suitable emulsifiers will depend in part on the composition and the final product.

The nutritional compositions of the present disclosure may optionally include one or more preservatives, which may also be added to extend the shelf life of the product. Suitable preservatives include, but are not limited to, potassium sorbate, sodium sorbate, potassium benzoate, sodium benzoate, calcium disodium EDTA, and mixtures thereof.

The nutritional compositions of the present disclosure may optionally include one or more stabilizers. Suitable stabilizers for use in practicing the nutritional compositions of the present disclosure include, but are not limited to, gum arabic, gum ghatti, gum karaya, gum tragacanth, agar, furcellaran, guar gum, gellan gum, locust bean gum, pectin, low methoxyl pectin, gelatin, microcrystalline cellulose, CMC (sodium carboxymethylcellulose), methylcellulose hydroxypropyl methylcellulose, hydroxypropyl cellulose, DATEM (diacetyl tartrate of mono-or diglycerides), dextrose, carrageenan, and mixtures thereof.

The nutritional compositions of the present disclosure may provide minimal, partial, or complete nutritional support. The composition may be a nutritional supplement or a meal replacement. The composition may be, but need not be, nutritionally complete. In one embodiment, the nutritional compositions of the present disclosure are nutritionally complete and contain the appropriate types and amounts of lipids, carbohydrates, proteins, vitamins, and minerals. The amount of lipid or fat can generally vary from about 2 to about 7 g/100 kcal. The amount of protein can generally vary from about 1 to about 5 g/100 kcal. The amount of carbohydrate can generally vary from about 8 to about 14 g/100 kcal.

In some embodiments, the nutritional composition of the present disclosure is growth-stage dairy. Growth-stage milk is a fortified milk-based beverage intended for children over the age of 1 year (typically 1-6 years of age). They are not medical foods and are not intended as meal replacers or supplements to address specific nutritional deficiencies. In contrast, long-term dairy products are designed for the purpose of acting as a supplement to diversify the diet in order to provide additional assurance that the child achieves a continuous daily intake of all essential vitamins and minerals, macronutrients, and additional functional dietary components, such as non-essential nutrients purported to have health promoting properties.

The aforementioned nutritional compositions are capable of promoting phase II enzyme gene expression in infants or children. In some embodiments, the composition further promotes and/or modulates phase II enzyme protein expression in infants and children. In some embodiments, the nutritional composition is designed to work with a conventional daily diet to support metabolic programming in pediatric subjects. The metabolic programming effect can be evident throughout childhood as well as adulthood, providing enhanced protection against harmful xenobiotics throughout the life of the subject. The aforementioned phase II enzyme can be, but is not limited to, glutathione transferase, UDP-glucuronyl transferase, NAD (P) H: quinone oxidoreductase 1, sulfotransferase, N- & O-methyltransferase, or mixtures thereof.

The exact composition of infant formula or growth-stage milk or other nutritional compositions according to the present disclosure may vary from market to market, depending on local regulations and dietary intake information for the target population. In some embodiments, the nutritional compositions according to the present disclosure consist of a milk protein source, such as whole or skim milk, with added sugar and sweeteners to achieve the desired organoleptic properties, and added vitamins and minerals. The fat composition is typically derived from a dairy-based material. The total protein target may be matched to human milk, cow milk, or lower values. The total carbohydrate is generally targeted to provide as little added sugar as possible, such as sucrose or fructose, to achieve an acceptable taste. Typically, vitamin a, calcium and vitamin D are added at levels that match the nutritional effects of regional cow's milk. Additionally, in some embodiments, vitamins and minerals are added at a level that provides about 20% of the Dietary Reference Intake (DRI) or 20% of the daily intake (DV) per serving. In addition, nutritional values may vary from market to market, depending on nutritional needs, raw material usage, and regional regulations established for the target population.

In one embodiment, the present disclosure relates to a supplement comprising phytonutrients for a pregnant or lactating female, wherein the supplement promotes phase II enzyme gene expression in a prenatal infant of the pregnant female or in an infant nursing from a lactating female. The phytonutrients may comprise any of the foregoing phytonutrients and mixtures thereof. In one embodiment, the supplement, when administered to a lactating or pregnant female, is capable of promoting phase II enzyme gene expression in an infant nursing from the lactating female or in a prenatal infant of the pregnant female. Exemplary phase II enzymes include, but are not limited to, GST, UGT, NQO1, sulfotransferase, N- & O-methyltransferase, and mixtures thereof. The supplement may further be administered to a female who may be pregnant.

In one embodiment, the supplement for pregnant and lactating women further comprises any additional nutrients, including vitamins, minerals, and fatty acids, which may be used to promote the health of the pregnant or lactating women and their infants. In an embodiment of the invention, the prenatal dietary supplement contains between about 0.1 and 10 mg folic acid. In another embodiment of the invention, the prenatal dietary supplement contains between about 0.3 and 5 mg folic acid. In particular embodiments, the prenatal dietary supplement contains between about 0.4 and 1mg folic acid. In yet another embodiment, the prenatal dietary supplement contains between about 400 and 700 μ g folic acid per day. In particular embodiments, the prenatal dietary supplement contains about 600 μ g per day of folic acid.

The prenatal dietary supplement of the present invention may be administered in one or more doses per day. In some embodiments, the prenatal dietary supplement is administered in two doses per day. In a separate embodiment, the prenatal dietary supplement is administered in three doses per day.

Any orally acceptable dosage form is contemplated by the present invention. Examples of such dosage forms include, but are not limited to, pills, tablets, capsules, liquids, liquid concentrates, powders, elixirs, solutions, suspensions, emulsions, lozenges, beads, cachets, and combinations thereof. Alternatively, the prenatal dietary supplement of the present invention may be added to a more complete nutritional product. In this embodiment, the nutritional product may contain protein, fat and carbohydrate components and may be used to supplement a diet or to serve as the sole source of nutrition.

The disclosure also provides metabolic programming methods for phase II enzymes. In one embodiment, the present disclosure relates to a method of promoting phase II enzyme gene expression in a pediatric subject comprising administering to the subject a nutritional composition comprising an effective amount of a phytonutrient. In one embodiment, the method further promotes phase II enzyme protein expression in a pediatric subject. The phytonutrients administered to the subject may include any of the foregoing phytonutrients and combinations thereof. In one embodiment, the method provides enhanced protection against xenobiotics during childhood, when the pediatric subject becomes an adult, or both. The phase II enzyme may be selected from the group consisting of glutathione transferase, UDP-glucuronyl transferase, NAD (P) H: quinone oxidoreductase 1, sulfotransferase, N- & O-methyltransferase, and mixtures thereof, but is not limited thereto.

The pediatric subject may be a child or an infant. For example, the subject may be an infant with an age ranging from 0 to 3 months, about 0 to 6 months, 0 to 12 months, 3 to 6 months, or 6 to 12 months. The subject may alternatively be a child with an age ranging from 1 to 13 years, 1 to 6 years, or 1 to 3 years. In one embodiment, the composition may be administered to the pediatric subject prenatally, during infancy, and during childhood.

The present disclosure also provides a method of promoting phase II enzyme gene expression in an infant nursing from a lactating female, comprising administering an effective amount of a phytonutrient to the lactating female, and feeding the infant with milk from the lactating female. In one embodiment, the method further promotes phase II enzyme protein expression. The infant may be suckled directly by the lactating female, or the milk of the lactating female may be expressed and then administered to the infant.

The present disclosure also provides a method of promoting phase II enzyme gene expression in a prenatal infant comprising administering to a female pregnant with the infant an effective amount of a phytonutrient. In another embodiment, the present disclosure provides a method of promoting phase II enzyme protein expression in a prenatal infant, comprising administering to a female pregnant with the infant an effective amount of a phytonutrient.

Phase II enzymes in the metabolic programming process include, but are not limited to, glutathione transferase, UDP-glucuronosyltransferase, NAD (P) H: quinone reductase, sulfotransferase, N- & O-methyltransferase, and mixtures thereof.

While not being bound by any particular theory, it is believed that the methods of the present invention provide metabolic programming in infants and children that will advantageously improve the ability of the subject to metabolize xenobiotics for the lifetime. For example, by promoting phase II enzyme gene and/or protein expression early in life, e.g., prenatal, infantile and childhood, the subject may have enhanced protection against potentially harmful xenobiotics later in life throughout childhood and adult life. Thus, improved metabolism and the ability to clear xenobiotics will provide enhanced protection against diseases and conditions regulated by harmful xenobiotics.

In one embodiment, the phytonutrient is selected from the group consisting of a polyphenol, a carotenoid, an isothiocyanate, or a mixture thereof. The polyphenol may be selected from, but is not limited to, flavonols, flavanols, flavanones, chalcones, flavonoids, isoflavonoids, anthocyanins, proanthocyanidins, anthocyanidins, and mixtures thereof. The carotenoid may be selected from, but is not limited to, lutein, zeaxanthin, astaxanthin, lycopene, beta-carotene, alpha-carotene, gamma-carotene, alpha-cryptoxanthin, beta-cryptoxanthin, and mixtures thereof. The isothiocyanate may be selected from, but is not limited to, sulforaphane, phenethylisothiocyanate, and mixtures thereof. In certain embodiments, the nutritional composition comprises between about 0.01 and about 70 mg isothiocyanate per liter. In some embodiments, the plant nutrient source does not comprise soy. In yet further embodiments, the plant nutrient is not soy isoflavonoid.

In one embodiment, the nutritional composition comprises from about 50 to about 1300nmol/L of a plant nutrient. In some embodiments, the nutritional composition comprises between about 0.01 and about 700 nmol/L of a plant nutrient. Further, the nutritional composition may include catechins in an amount of from about 500 to about 2000 nmol/L, or quercetin in an amount of from about 50 to about 400 nmol/L. In another embodiment, the composition provides about 5 to 50 mg/d of sulforaphane. In certain embodiments, the composition comprises between 0.01 and 70 mg/L of at least one isothiocyanate, such as sulforaphane. In some embodiments, the nutritional composition is formulated to deliver between about 0.01 and about 300 mg/d of the plant nutrient component. In some embodiments, the nutritional composition is formulated to deliver between about 0.01 and about 170 mg/d flavan-3-ols, such as catechins, and in certain embodiments, the nutritional composition is formulated to deliver between about 0.01 and about 150 mg/d flavonols, such as quercetin.

The examples are provided to illustrate some embodiments of the nutritional compositions of the present disclosure, but should not be construed as limiting in any way. Other embodiments within the scope of the claims herein will be apparent to those skilled in the art from consideration of the specification or practice of the nutritional compositions or methods disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the disclosure being indicated by the claims which follow the examples.

Examples

Effect of phytonutrients on phase II enzyme expression in human primary dermal fibroblasts

Materials: sulforaphane (4-methylsulphinylbutyl isothiocyanate; purity 98%) was purchased from LKT laboratories (Alexis biochemicals, UK), while catechin and quercetin were purchased from Sigma (UK). For cytotoxicity assays of cells, WST-1 reagent was obtained from roche (uk), while for quantitative PCR, the Bioscript RT kit, random hexamer, RNase Out inhibitor and master mix kit were purchased from Bioline, Promega, Invitrogen and Primer Design (Primer Design), respectively. Rabbit polyclonal GSTA1 was obtained from Calbiochem and goat polyclonal NQO1 and UGT1A was obtained from Santa Cruz. All other materials and reagents were purchased from Sigma Aldrich, UK unless otherwise noted.

Cell culture: normal primary human dermal fibroblasts at 1 month age (CCD-32sk), 2 months age (CCD-1092sk), and adult (142Br) were obtained from ATCC and ECACC. All cells were incubated at 37 ℃ with 5% CO₂Cultured in MEM under humidified atmosphere with GlutaMAX-1(GIBCO) medium supplemented with 10% FBS (V/V), 1% antibiotics, and 1% NEAA (GIBCO). The cell culture medium was changed every 48 hours, or subcultured appropriately, and used within 10 passages.

Cytotoxicity assay cytotoxicity of cells was assessed by a WST-1 assay that measures mitochondrial dehydrogenase activity following phytochemical treatment the dehydrogenase of living cells cleaved tetrazolium salts to produce formazan and detected a change in absorbance briefly, cells were labeled 2 × 10⁴The/well was seeded on 96-well plates and allowed to attach overnight. Then, treatment with 5, 10, 25, 50 or 100 μ M sulforaphane, catechin or quercetin was performed for 24 hours, plus no treatment control. At the end of the treatment period, 10 μ l of WST-1 reagent was added to each well, and the plates were incubated at 37 ℃, 95% air, 5% CO₂Is incubated for 2 hours under a humidified atmosphere. Absorbance at 450nm was measured and the average of three blank wells containing medium and WST-1 reagent only was subtracted from each absorbance reading. The resulting values were used for data analysis.

RNA extraction and analysis by TaqMan real-time PCR: total cellular RNA was isolated using the Genelute Total mammalian RNA kit (Sigma-Aldrich) following the manufacturer's instructions. Total RNA (260/280nm scale) was quantified using a NanoDrop spectrophotometer (Labtech International, UK) and up to 1. mu.g of RNA was reverse transcribed using a Bioscript RT kit and random hexamers and RNase OUT inhibitor. Using ABI prism7500 sequence detection System (Applied Biosystems), mRNA expression was determined by TaqMan real-time PCR. PCR reactions were performed in 96-well plates with the master mix kit in a total volume of 25 μ Ι _ per well consisting of: suitable 1 or 5ng of sample, 100 nmol/L of probe labeled with 5 '-reporter dye FAM (6-carboxyfluorescein) and 3' -quencher TAMRA (6-carboxytetramethylrhodamine), and 200nmol/L of upstream and downstream primers. A standard curve was established with serial dilutions of the control sample and analyzed using ABI software. Data were normalized to housekeeping gene 18S ribosomal RNA. By passing^ΔΔCt method to quantify gene expression, with fold induction =2^{Δ Δ Ct (control) -Ct (treatment)}(Livak, K.J., Schmittgen ,T.D., Analysis of relativegene expression data using real-time quantitative PCR and the 2(-Delta DeltaC(T)) Method.Methods2001. 25, 402-408)。

Preparation and immunoblotting of protein extracts: treated and control cells were washed twice with ice cold Phosphate Buffer (PBS) and subsequently incubated in Nonidet P-40(NP-40) buffer (20mM Tris-HCl, pH 8, 150mM NaCl, 10% glycerol, 1% NP-40) for 30 minutes, containing an intact mini-EDTA-free protease inhibitor cocktail (Complete mini-EDTA-free protease inhibitor cocktail, Roche) in 10 ml buffer. Cells were harvested by peeling and the homogenate was centrifuged at 13,684g, 4 ℃ for 15 minutes. The supernatant was collected and frozen at-80 ℃. Protein concentration was determined using Bradford reagent (Sigma) following the manufacturer's instructions. Mu.g of the protein lysate were separated on a 10% SDS-polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane (Bio-Rad) using a semi-dry transfer cell (Trans-Blot; Bio-Rad). The membranes were blocked with Marvel skim milk powder in PBS (5% w/v), Tween 20 (0.05%, v/v) for 1 hour at room temperature or overnight at 4 ℃. The target protein was visualized by contacting the membrane with the first antibody in milk for 2 hours at room temperature. Dilutions of the antibody were rabbit polyclonal GSTA1, 1:2000, goat polyclonal NQO 11: 1000, and goat polyclonal UGT1A 1: 1000. Following primary antibody incubation, the membrane was incubated with the appropriate HRP-conjugated secondary antibody and the signal detected using the enhanced chemiluminescence kit (GE Healthcare) following the manufacturer's instructions. β -actin levels were determined as loading controls and bands were visualized using a Fujifilm LAS3000 Imager.

Statistical analysis: statistical analysis was performed with statistical software SPSS (version 13.1). To evaluate the effect of the various treatments, a one-way ANOVA followed by a post-test T-test was used. If it is notP<0.05, the difference is considered significant. Unless otherwise stated, results are expressed as mean plus SEM of three separate runs.

Results

Cytotoxicity of quercetin, catechin and sulforaphane: the effect of quercetin, catechin and sulforaphane on phase II enzyme expression was evaluated using an in vitro cell model of early infancy (1 month or 2 years of age) and a comparable adult model. A series of phytochemical concentrations (1-100 μ M) were applied to each cell model to examine dose-response and determine the optimal concentration for subsequent experiments. WST-1 cell viability experiments demonstrated that cells isolated from 1 month old (fig. 1A) and adult (fig. 1C) donors tolerated all candidate phytochemicals at concentrations up to 50 μ M, and viability was at least 80%. Cells isolated from 2-year-old donors (fig. 1B) tolerated quercetin and catechin concentrations up to 50 μ M, and survivability was at least 80%. However, incubation with sulforaphane at concentrations ranging from 50 and 100 μ M induced significant cell death. Based on these data, subsequent experiments were performed at 5, 10 and 20 μ M to avoid the toxic effects of high phytochemical doses on the cells.

Quercetin differentially affected mRNA phase II enzymes in a1 month old cell model: cells from the 1-month-old model incubated with quercetin (fig. 2A black bars) showed significant dose-response up-regulation of GST (3.9-fold) and NQO1mRNA (7.2-fold) compared to the control group (P < 0.05). Similarly, and at the highest quercetin concentration (20 μ M), cells from the adult cell model also showed upregulation of GST (7.4-fold) and UGT (5.5-fold) (FIG. 2A blank bar). In contrast, cells obtained from the 2-year-old cell model did not show any significant change in expression of GST, UGT, or NQO1mRNA at all concentrations of quercetin tested (fig. 2A grey bars).

GST and NQO1mRNA expression in infant cell lines were significantly upregulated in response to catechins: cells from the adult cell model (fig. 2B blank bars) showed dose-response upregulation in response to catechin-treated GST and NQO1mRNA, and UGT also showed an increase. The 1 month old cell model showed a significant increase (3.7-fold) in GST mRNA, but expression of NQO1 and UGT mRNA was unaffected. The 2-year-old cell model showed a significant increase (4.5-fold) in NQO1 expression after catechin treatment.

The infant cell lines showed significantly increased NQO1mRNA expression following sulforaphane treatment: cells from the adult cell model (fig. 2C blank bars) showed significant increases in GST, UGT, and NQO1mRNA levels; in the infant cell model, particularly after sulforaphane treatment, a significant increase (6-35 fold increase) in NQO1 was observed compared to the control group (fig. 2C black and gray bars). In the infant cell model, expression of GST mRNA was also significantly elevated in response to sulforaphane, but not to the same extent as NQO 1.

Protein expression in infant cell models: in a 1-month-old cell model, expression of NQO1, UGT, and GST showed no significant response to three phytochemicals (except for sulforaphane), while sulforaphane showed significant induction of UGT expression (fig. 3A). In the 2-year-old cell model, phytochemicals induced the expression of all enzyme proteins except 10 μ M quercetin. In addition, the expression of UGT, GST and NQO1 proteins was significantly upregulated in response to higher doses of catechin and sulforaphane (fig. 3B). In an adult cell model, it was observed that NQO1 protein expression was significantly upregulated in response to sulforaphane and higher doses of quercetin and catechin treatment (fig. 3C). UGT expression was affected by sulforaphane treatment, while GST expression was elevated in response to quercetin and catechin (fig. 3C).

All references cited in this specification, including, but not limited to, all papers, publications, patents, patent applications, presentations, texts, reports, manuscripts, manuals, books, web postings, journal articles, periodicals, and the like, are incorporated herein by reference in their entirety. The discussion of the references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinency of the cited references.

Although embodiments of the present disclosure have been described using specific terms, devices, and methods, such description is for illustrative purposes only. The words used are words of description rather than of limitation. It is to be understood that modifications and variations may be resorted to by those of ordinary skill in the art without departing from the spirit or scope of this disclosure as set forth in the following claims. Further, it should be understood that aspects of the various embodiments may be interchanged both in whole or in part. For example, while the production of commercially sterile liquid nutritional supplements prepared according to these methods have been exemplified, other uses are envisioned. Therefore, the spirit and scope of the appended claims should not be limited to the versions of the specification contained herein.

Claims (50)

1. Use of a phytonutrient in the manufacture of a nutritional composition for promoting phase II enzyme gene expression in a pediatric subject, wherein the phytonutrient is selected from a polyphenol, a carotenoid, an isothiocyanate, or a mixture thereof, and wherein the nutritional composition comprises from 50 to 1300nmol/L of the phytonutrient.

2. The use of claim 1, wherein the nutritional composition further promotes phase II enzyme protein expression.

3. The use of claim 1, wherein the nutritional composition provides enhanced protection against xenobiotics when the pediatric subject is an adult.

4. The use of claim 1, wherein the phase II enzyme is selected from the group consisting of glutathione transferase, UDP-glucuronyl transferase, NAD (P) H: quinone oxidoreductase 1, sulfotransferase, N- & O-methyltransferase, and mixtures thereof.

5. The use of claim 1, wherein the pediatric subject is an infant with an age ranging from 0 to 12 months.

6. The use of claim 1, wherein the pediatric subject is a child ranging in age from 1 to 6 years.

7. The use of claim 1, wherein the nutritional composition is administered to the pediatric subject prenatally, during infancy, and during childhood.

8. The use of claim 1, wherein the polyphenol comprises a flavonoid.

9. The use of claim 1, wherein the carotenoid is selected from the group consisting of lutein, zeaxanthin, astaxanthin, lycopene, beta-carotene, alpha-carotene, gamma-carotene, alpha-cryptoxanthin, beta-cryptoxanthin, and mixtures thereof.

10. The use of claim 1, wherein the isothiocyanate is selected from the group consisting of sulforaphane, phenethylisothiocyanate, and mixtures thereof.

11. The use of claim 1, wherein the nutritional composition comprises from 500 to 1300nmol/L of catechins.

12. The use of claim 1, wherein the composition comprises 50 to 400 nmol/L of quercetin.

13. The use of claim 1, wherein the composition comprises sulforaphane.

14. Use of an effective amount of a phytonutrient in the manufacture of a nutritional composition for promoting phase II enzyme gene expression in an infant nursing from a lactating female, wherein the nutritional composition is administered to the lactating female, wherein the phytonutrient is present in the nutritional composition in an amount of 50 to 1300nmol/L and is selected from a polyphenol, a carotenoid, an isothiocyanate, or a mixture thereof.

15. The use of claim 14, wherein the nutritional composition further promotes phase II enzyme protein expression.

16. The use of claim 14, wherein the nutritional composition provides enhanced protection against xenobiotics when the infant becomes an adult.

17. The use of claim 14, wherein the phase II enzyme is selected from the group consisting of glutathione transferase, UDP-glucuronyltransferase, nad (p) H: quinone oxidoreductase 1, sulfotransferase, N- & O-methyltransferase, and mixtures thereof.

18. Use of a phytonutrient for the manufacture of a nutritional composition for promoting phase II enzyme gene expression in a prenatal infant, wherein the nutritional composition is administered to a female pregnant with the prenatal infant, and wherein the phytonutrient is selected from a polyphenol, a carotenoid, an isothiocyanate, or a mixture thereof, and wherein the nutritional composition comprises from 50 to 1300nmol/L of the phytonutrient.

19. The use of claim 18, wherein the nutritional composition further promotes phase II enzyme protein expression.

20. The use of claim 18, wherein the nutritional composition provides enhanced protection against xenobiotics when the prenatal infant becomes an adult.

21. The use of claim 18, wherein the phase II enzyme is selected from the group consisting of glutathione transferase, UDP-glucuronyltransferase, nad (p) H: quinone oxidoreductase 1, sulfotransferase, N- & O-methyltransferase, and mixtures thereof.

22. A milk-based nutritional composition comprising a fat source, a carbohydrate source, a protein source, and 50 to 1300nmol/L of a phytonutrient source, wherein the composition is capable of promoting phase II enzyme gene expression in a pediatric subject, and wherein the phytonutrient source is selected from a polyphenol, a carotenoid, an isothiocyanate, or a mixture thereof.

23. The milk-based nutritional composition of claim 22, wherein the composition further promotes phase II enzyme protein expression.

24. The milk-based nutritional composition of claim 22, wherein the phase II enzyme is selected from the group consisting of glutathione transferase, UDP-glucuronyl transferase, nad (p) H: quinone oxidoreductase 1, sulfotransferase, N- & O-methyltransferase, and mixtures thereof.

25. The milk-based nutritional composition of claim 22, wherein the pediatric subject is an infant from 0 to 12 months.

26. The milk-based nutritional composition of claim 22, wherein the pediatric subject is a child of 1 to 6 years of age.

27. The milk-based nutritional composition of claim 22, wherein the polyphenol comprises a flavonoid.

28. The milk-based nutritional composition of claim 22, wherein the carotenoid is selected from the group consisting of lutein, zeaxanthin, astaxanthin, lycopene, beta-carotene, alpha-carotene, gamma-carotene, alpha-cryptoxanthin, beta-cryptoxanthin, and mixtures thereof.

29. The milk-based nutritional composition of claim 22, wherein the isothiocyanate is selected from the group consisting of sulforaphane, phenethylisothiocyanate, and mixtures thereof.

30. The milk-based nutritional composition of claim 22, wherein the composition comprises from 500 to 1300nmol/L catechins.

31. The milk-based nutritional composition of claim 22, wherein the composition comprises 50 to 400 nmol/L of quercetin.

32. The milk-based nutritional composition of claim 22, wherein the composition comprises sulforaphane.

33. The milk-based nutritional composition of claim 22, further comprising a source of long chain polyunsaturated fatty acids.

34. The milk-based nutritional composition of claim 33, wherein the source of long chain polyunsaturated fatty acids comprises docosahexaenoic acid, arachidonic acid, or a mixture thereof.

35. The milk-based nutritional composition of claim 22, further comprising a prebiotic.

36. The milk-based nutritional composition of claim 35, wherein the prebiotic is polydextrose, galactooligosaccharide, or a mixture thereof.

37. The milk-based nutritional composition of claim 22, further comprising beta-glucan.

38. The milk-based nutritional composition of claim 22, further comprising a viable probiotic or a killed probiotic.

39. A supplement for pregnant or lactating women comprising a source of phytonutrients of 50 to 1300nmol/L, wherein the supplement is capable of promoting expression of a phase II enzyme gene in:

a) the prenatal infant of said pregnant female, or

b) An infant suckled by said lactating woman, and

wherein the phytonutrient source is selected from the group consisting of polyphenols, carotenoids, isothiocyanates, or mixtures thereof.

40. The supplement of claim 39, wherein the supplement is further capable of promoting phase II enzyme protein expression.

41. The supplement of claim 39, wherein the phase II enzyme is selected from the group consisting of glutathione transferase, UDP-glucuronyl transferase, NAD (P) H: quinone oxidoreductase 1, sulfotransferase, N- & O-methyltransferase, and mixtures thereof.

42. The supplement of claim 39, wherein the polyphenol comprises a flavonoid.

43. The supplement of claim 39, wherein the carotenoid is selected from the group consisting of lutein, zeaxanthin, astaxanthin, lycopene, beta-carotene, alpha-carotene, gamma-carotene, alpha-cryptoxanthin, beta-cryptoxanthin, and mixtures thereof.

44. The supplement of claim 39, wherein the isothiocyanate is selected from the group consisting of sulforaphane, phenethylisothiocyanate, and mixtures thereof.

45. The use of claim 1, wherein the polyphenol is selected from the group consisting of flavonols, flavanols, flavones, isoflavonoids, anthocyanins, proanthocyanidins, anthocyanidins, and mixtures thereof.

46. The milk-based nutritional composition of claim 22, wherein the polyphenol is selected from the group consisting of flavonols, flavanols, flavones, isoflavonoids, anthocyanins, proanthocyanidins, anthocyanidins, and mixtures thereof.

47. The supplement of claim 39, wherein the polyphenol is selected from the group consisting of flavonols, flavanols, flavones, isoflavonoids, anthocyanins, proanthocyanidins, anthocyanidins, and mixtures thereof.

48. The use of claim 45, wherein the polyphenol is flavan-3-ol.

49. The milk-based nutritional composition of claim 46, wherein the polyphenol is flavan-3-ol.

50. The supplement of claim 47, wherein the polyphenol is flavan-3-ol.