Compositions for increasing the level of NQO-1 or its gene expression

Abstract

To provide compositions for increasing the level of NAD(P)H:quinone oxidoreductase 1 (NQO-1), or increasing its gene expression.SOLUTION: Provided is a composition for increasing the level of NAD(P)H:quinone oxidoreductase 1 (NQO-1) or increasing its gene expression in a subject. The composition comprises a combination of sulforaphane and a glucan as active ingredients, which combine to produce a synergistic effect between the active ingredients. The glucan is provided as a Ganoderma lucidum extract or powder. The Ganoderma lucidum extract preferably contains about 1 to about 75 wt.% of one or more glucans. Furthermore, the one or more glucans are preferably β-glucan and / or 1,3-β-glucan and / or 1,6-β-glucan.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application claims priority to the following applications, each of which is incorporated by reference in its entirety: U.S. Provisional Patent Application No. 61 / 668,328, filed July 5, 2012; U.S. Provisional Patent Application No. 61 / 668,342, filed July 5, 2012; U.S. Provisional Patent Application No. 61 / 668,386, filed July 5, 2012; U.S. Provisional Patent Application No. 61 / 668,396, filed July 5, 2012; U.S. Provisional Patent Application No. 61 / 668,364, filed July 5, 2012; U.S. Provisional Patent Application No. 61,668,374, filed July 5, 2012; and U.S. Provisional Patent Application No. 61 / 794,417, filed March 15, 2013.

[0002] The present invention relates to a combination of a sulforaphane precursor, an enzyme capable of converting the sulforaphane precursor to sulforaphane, an enzyme enhancer, and a mushroom (preferably, Maitake, Shiitake, or Reishi) extract or powder. The present invention also relates to a combination of sulforaphane or a derivative thereof and a mushroom (preferably, Maitake, Shiitake, or Reishi) extract or powder. The present invention also relates to a combination of a broccoli extract or powder and a mushroom (preferably, Maitake, Shiitake, or Reishi) extract or powder. The present invention provides compositions and methods related to these combinations. [Background technology]

[0003] The use of natural products is becoming increasingly popular for humans and companion animals. Some of these natural products are incorporated into dietary supplements and medical foods. There is a need in the art for dietary supplements that are useful as chemoprotective agents and / or antioxidants. Additionally, there is a need in the art for pharmaceutical compositions and dietary supplements that are useful for breast-related conditions and disorders.

[0004] Chemoprotection through the use of natural products is developing as a safe, effective, inexpensive, readily available, and practical means to prevent or reduce the occurrence of numerous conditions affecting humans and livestock. It has been found that carcinogens, which can damage cells at the molecular level, are often ingested and inhaled as non-toxic precursors. These non-toxic precursors can then be converted into carcinogens in the body. Chemoprotectants, such as natural substances that can activate detoxification enzymes or their cofactors, can counteract, allow elimination, or enhance other naturally occurring defenses, such as the immune system.

[0005] Some natural products have antioxidant activity. Oxidative stress plays a major role in aging, the progression of neurodegenerative diseases, and physiological trauma such as ischemia. Antioxidants can reduce or inhibit the oxidation of important biomolecules, and can play a role in treating, preventing, or reducing the occurrence of cancer, coronary heart disease, stroke, and neurodegenerative diseases, and Alzheimer's disease, dementia, and stroke are examples of conditions affected by oxidative stress.

[0006] Cancer is thought to be primarily the result of exposure to environmental insults, whether internal (i.e., estrogen, progesterone hormones) or external (i.e., bisphenol A (BPA) from plastics), and chronic inflammation. Fortunately, damage from environmental insults can be counteracted by a complex network of phase II chemoprotective enzymes found in numerous cell types throughout the body. It is well known that estrogen and its metabolites can lead to breast tissue and tumor growth. To make matters worse, quinone estrogen metabolites have the ability to invade breast tissue and migrate into the nuclei of ductal and glandular epithelial cells. There, they bind to DNA to form quinone estrogen-DNA adducts, leading to downstream mutations. These mutations are thought to be involved in cancer initiation, the very basis of tumor development. Fortunately, a specific phase II enzyme, NAD(P)H:quinone oxidoreductase (NQO1), can capture dangerous, highly reactive quinone estrogens and metabolize them into inactive chemicals that can be easily eliminated from the body. Therefore, the main mechanism for reducing cancer incidence is to induce protective phase II enzymes, including NQO1. Increasing the level of NQO1 can be effective in treating, preventing, repairing, reducing the occurrence and decreasing symptoms associated with any condition caused by high levels of quinone estrogens. Examples of quinone estrogens include, but are not limited to, estrogenic catechol quinones. Quinone estrogens are described in the following references, each of which is incorporated by reference in its entirety: Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3; and Non-Patent Document 4.

[0007] One example of a natural product thought to have chemoprotective and antioxidant properties is sulforaphane. Sulforaphane is an organosulfur compound also known as 1-isothiocyanato-4-methylsulfinylbutane. The sulforaphane precursor, glucoraphanin, can be obtained from cruciferous vegetables such as broccoli, Brussels sprouts, and cabbage. However, to obtain levels adequate for chemoprevention, large amounts of vegetables must be consumed. Glucoraphanin is converted to sulforaphane by a thioglucosidase enzyme called myrosinase, which occurs endogenously in various exogenous sources, such as cruciferous vegetables, and in the gut microbiota. However, upon oral ingestion of glucoraphanin, not all animals are able to achieve its conversion to sulforaphane, most likely due to variations in the microbiota population and overall health. Furthermore, in acidic environments such as the stomach, glucoraphanin can be converted to inactive metabolites. The active metabolite, sulforaphane, induces nuclear erythroid-2-related factor (Nrf2), which then upregulates the production of phase II detoxification and cellular defense enzymes, such as glutathione S-transferase, NAD(P)H:quinone oxidoreductase (NQO1), and heme oxygenase-1 (HO-1). Sulforaphane is thought to induce the production of these enzymes without significantly altering the synthesis of P-450 cytochrome enzymes. Upregulation of phase II enzymes is thought to play a role in various vital processes, including protecting the brain from cytotoxicity, protecting the liver from the toxic effects of fat accumulation, and detoxifying various other tissues.

[0008] Sulforaphane and its precursor glucoraphanin have been widely studied. Shapiro et al. (Non-Patent Document 5) discuss a Phase I clinical study to determine the safety, tolerability, and metabolism of broccoli sprout glucosinolates and isothiocyanates. Shapiro et al. discuss a placebo-controlled, double-blind, randomized clinical trial of sprout extracts containing either glucosinolates such as glucoraphanin or isothiocyanates such as sulforaphane in healthy human subjects. The study found that administration of these substances did not result in systematic, clinically significant side effects. Ye et al. (Non-Patent Document 6) discuss the pharmacokinetics of broccoli sprout isiothiocyanates in humans.

[0009] Several mushrooms have been used or studied for their medicinal properties. These "medicinal mushrooms" are believed to have beneficial properties, such as antiviral, antimicrobial, anticancer, hypoglycemic, and / or anti-inflammatory activities. Examples of medicinal mushrooms include maitake, shiitake, reishi, cremini, almond, chestnut, wood ear, cloud ear, porcini, ink cap, yarta gunbu, enokitake, shimeji, tiger milk, morel, bamboo, tamogitake, pink oyster, king oyster mushroom, oyster mushroom, cauliflower, white wood ear, golden jelly, matsutake, Mexican truffle, and straw mushroom.

[0010] Maitake mushrooms (Grifola frondosa) are widely consumed as food and are used in traditional medicine to enhance immune function and treat cancer. Maitake mushrooms, which contain glucans, are believed to have beneficial properties, including antitumor and immunomodulatory effects. Standardized extracts derived from maitake mushrooms contain glucans, such as protein-linked β-glucans, as active ingredients. β1,6-glucan (a protein-linked polysaccharide) has been identified as the active ingredient in maitake mushrooms. Maitake mushrooms have been demonstrated to have antitumor effects, inhibiting tumor metastasis in vitro. In one study, tumor regression or significant symptom improvement was observed in half of the subjects using maitake extract. A study of postmenopausal breast cancer patients showed that oral administration of maitake extract had immunomodulatory effects.

[0011] Shiitake mushrooms (Lentinula edodes) are edible mushrooms native to East Asia. Shiitake mushrooms contain mycochemicals hypothesized to have antiviral, antibiotic, anti-inflammatory, antihypertensive, and anticarcinogenic effects. This is thought to be largely the result of glucans, both α- and β-glucans. Some shiitake mushroom extracts have an α-glucan content of over 40%. Furthermore, lentinan (1,3β-D-glucan, a polysaccharide isolated from shiitake mushrooms) has been well studied and is thought to play a role in shiitake's beneficial effects. It has been shown to have anticancer effects in colon cancer cells, which may be due to its ability to inhibit the cytochrome P450-1A enzyme, which is known to metabolize carcinogen precursors to their active forms. Lentin (its protein component) has potent antifungal properties, inhibits leukemia cell growth, and suppresses the activity of human immunodeficiency virus-1 reverse transcriptase.

[0012] Reishi mushroom (Ganoderma lucidum), also known as lingzki mushroom, is an edible mushroom found in East Asia. Reishi mushrooms are thought to have antitumor, anticancer, immunomodulatory, and immunotherapeutic effects. Reishi mushrooms possess several components that are thought to contribute to their activity, including glucans such as β-glucan, canthaxanthin, sterols, coumarin, ganoderic acid, and mannitol.

[0013] Baker's yeast (Saccaromyces cerevisiae) can be a source of glucans, particularly β-glucans. The active components of baker's yeast can be extracted by several methods, such as those described in Non-Patent Document 7, Patent Document 1, Patent Document 2, and Patent Document 3, each of which is incorporated by reference in its entirety.

[0014] Glucans are described in the following references, each of which is incorporated by reference in its entirety: Glucans, 1999, pp. 111-114, 1999; and 1999, pp. 111-114, 1999.

[0015] Zhang et al. (Non-Patent Document 10) discuss a study in Sprague-Dawley rats to determine the anticarcinogenic activity of sulforaphane and structurally related synthetic norbornyl isiothiocyanates. The study determined that administration of sulforaphane was effective in preventing the formation of mammary tumors.

[0016] Cornblatt et al. (Non-Patent Document 11) discuss a study in Sprague-Dawley rats to determine the effect of sulforaphane in chemoprevention in the breast. The study determined that oral administration of either sulforaphane resulted in a three-fold increase in NAD(P)H:quinone oxidoreductase (NQO1) enzyme activity and a four-fold increase in immunostaining for the heme oxygenase-1 (HO-1) enzyme in the mammary epithelium.

[0017] Munday et al. (Non-Patent Document 12) discuss a study on the effect of a freeze-dried aqueous extract of broccoli sprouts on bladder carcinogenesis in rats. The study found that administration of broccoli sprout extract resulted in significant induction of glutathione S-transferase and NAD(P)H:quinone oxidoreductase 1 in the bladder, enzymes with protective activity against oxidants and carcinogens.

[0018] Fang et al. (Non-Patent Document 13) disclosed a study to determine the antiproliferative effects of the ethyl acetate fraction of Lentinula edodes mushroom on human breast carcinoma cell lines (MDA-MB-453 and MCF-7), a human non-malignant breast epithelial cell line (MCF-10F), and two myeloma cell lines (RPMI08226 and IM-9). The study found that the inhibition of tumor cell growth by components in Lentinula edodes mushroom could be attributed to the induction of apoptosis.

[0019] Kim et al. (Non-Patent Document 14) have disclosed a study investigating the natural killer (NK) cell activation and anti-cancer effects of rice bran-derived exobiopolymers cultured from shiitake mushrooms (Lentinus edodes). This study found that exobiopolymers may be effective for the prevention and / or treatment of cancer through natural killer cell activation.

[0020] Louie et al. (Non-Patent Document 15) discuss the synergistic effect of a combination of interferon-α and Maitake mushroom D-fraction (PDF, a bioactive mushroom extract) on the anticancer activity of interferon-α in bladder cancer T24 cells in vitro.

[0021] Masuda et al. (Non-Patent Document 16) discuss a study evaluating the anti-metastatic activity of a fraction of Maitake mushroom in a mouse model of lung metastasis. The study found that the fraction inhibited tumor metastasis by inhibiting tumor cell adhesion to vascular endothelial cells through activation of natural killer cells and antigen-presenting cells (APCs) and suppression of adhesion molecules such as ICAM-1.

[0022] Patent Document 4 discloses a formulation containing an encapsulated or coated glucosinolate such as glucoraphanin and myrosinase.

[0023] All references cited herein are incorporated by reference in their entirety. [Prior art documents] [Patent documents]

[0024] [Patent Document 1] U.S. Patent No. 7,803,605 [Patent Document 2] U.S. Patent No. 5,702,719 [Patent Document 3] U.S. Patent No. 8,323,644 [Patent Document 4] European Patent Application No. 2213280 [Non-patent literature]

[0025] [Non-Patent Document 1] Nutter et al., Chem Res Toxicol, 1994, 7:23-28 [Non-patent document 2] Cavalieri et al., Ann NY Acad Sci, 2006;1089:286-301 [Non-patent document 3] Bolton et al., Chem Res Toxicol, 2008, 21(1):93-101 [Non-patent document 4] Cavalieri, Biochimica et Biophysica Acta, 2006, Volume 1766: Pages 63~78

Non-patented document 5

Non-patent document 6

Non-patent document 7

Non-patent document 8

[0026] The present invention provides a composition comprising (i) a sulforaphane precursor, preferably glucoraphanin; (ii) an enzyme capable of converting the sulforaphane precursor to sulforaphane, preferably a glucosidase enzyme, more preferably a thioglucosidase enzyme, and most preferably myrosinase; (iii) an enzyme enhancer, preferably ascorbic acid; and (iv) a mushroom (preferably, Maitake, Lentinula edodes, or Reishi mushroom) extract or powder. The present invention also provides a method for treating, preventing, reducing the incidence of, reducing symptoms associated with, and / or reducing secondary recurrence of cancer, particularly breast cancer, prostate cancer, colon cancer, lung cancer, and bladder cancer, in a subject, comprising administering to the subject (i) a sulforaphane precursor, (ii) an enzyme capable of converting the sulforaphane precursor to sulforaphane, (iii) an enzyme enhancer, and (iv) a mushroom (preferably, Maitake, Lentinula edodes, or Reishi mushroom) extract or powder. The present invention also provides a method for increasing the level of or increasing gene expression of NAD(P)H:quinone oxidoreductase 1 (NQO-1) in a subject, comprising administering to the subject (i) a sulforaphane precursor, (ii) an enzyme capable of converting the sulforaphane precursor to sulforaphane, (iii) an enzyme enhancer, and (iv) a mushroom (preferably, Maitake, Shiitake, or Reishi mushroom) extract or powder. The present invention also provides a method for treating, preventing, reducing the occurrence of, reducing symptoms associated with, and / or reducing secondary recurrence of a disease or condition associated with elevated levels of quinone estrogens, comprising administering to the subject (i) a sulforaphane precursor, (ii) an enzyme capable of converting the sulforaphane precursor to sulforaphane, (iii) an enzyme enhancer, and (iv) a mushroom (preferably, Maitake, Shiitake, or Reishi mushroom) extract or powder.

[0027] The present invention provides a composition comprising (i) sulforaphane or a derivative thereof and (ii) a mushroom (preferably, maitake, shiitake, or reishi mushroom) extract or powder. The present invention also provides a method for treating, preventing, reducing the incidence of, alleviating symptoms associated with, and / or reducing secondary recurrence of cancer, particularly breast cancer, prostate cancer, colon cancer, lung cancer, and bladder cancer, in a subject, comprising administering to the subject (i) sulforaphane or a derivative thereof and (ii) a mushroom (preferably, maitake, shiitake, or reishi mushroom) extract or powder. The present invention also provides a method for increasing the level of, or increasing gene expression of, NAD(P)H:quinone oxidoreductase 1 (NQO-1) in a subject, comprising administering to the subject (i) sulforaphane or a derivative thereof and (ii) a mushroom (preferably, maitake, shiitake, or reishi mushroom extract) or powder. The present invention also provides a method for treating, preventing, reducing the occurrence of, diminishing symptoms associated with, and / or reducing secondary recurrence of a disease or condition associated with elevated levels of quinone estrogens, comprising administering to a subject (i) sulforaphane or a derivative thereof and (ii) a mushroom (preferably, Maitake, Shiitake, or Reishi mushroom) extract or powder.

[0028] The present invention provides a composition comprising (i) a broccoli extract or powder and (ii) a mushroom (preferably, maitake, shiitake, or reishi mushroom) extract or powder. The present invention also provides a method for treating, preventing, reducing the incidence of, alleviating symptoms associated with, and / or reducing secondary recurrence of cancer, particularly breast cancer, prostate cancer, colon cancer, lung cancer, and bladder cancer, in a subject, comprising administering to the subject (i) a broccoli extract or powder and (ii) a mushroom (preferably, maitake, shiitake, or reishi mushroom) extract or powder. The present invention also provides a method for increasing the level of, or increasing gene expression of, NAD(P)H:quinone oxidoreductase 1 (NQO-1) in a subject, comprising administering to the subject (i) a broccoli extract or powder and (ii) a mushroom (preferably, maitake, shiitake, or reishi mushroom) extract or powder. The present invention also provides a method for treating, preventing, reducing the occurrence of, diminishing symptoms associated with, and / or reducing secondary recurrence of a disease or condition associated with elevated levels of quinone estrogens, comprising administering to a subject (i) a broccoli extract or powder and (ii) a mushroom (preferably, Maitake, Shiitake, or Reishi mushroom) extract or powder. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 is a graph showing the conversion of glucoraphanin at 38° C. without ascorbic acid, as described in Example 4. [Figure 2] 1 is a graph showing conversion within about 10 minutes at 38° C. as a function of ascorbic acid concentration, as described in Example 4. [Figure 3] 1 is a graph showing the conversion to sulforaphane within 30 minutes at 38° C. and 1 mM ascorbic acid, as described in Example 4. [Figure 4] 1 is a graph showing the conversion of glucoraphanin to sulforaphane in simulated intestinal fluid, as described in Example 5. [Figure 5] 1 is a graph showing the results of the experiment described in Example 6. [Figure 6] 1 is a graph showing the results of the experiment described in Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention relates to a combination of a sulforaphane precursor, an enzyme capable of converting the sulforaphane precursor to sulforaphane, an enzyme enhancer, and a mushroom (such as Maitake, Shiitake, or Reishi mushroom) extract or powder. The present invention also relates to a combination of sulforaphane or a derivative thereof and a mushroom (such as Maitake, Shiitake, or Reishi mushroom) extract or powder. The present invention also relates to a combination of a broccoli extract or powder and a Maitake, Shiitake, or Reishi mushroom extract or powder. The present invention also relates to the use of a mushroom extract or powder together with a mixture of one or more of a sulforaphane precursor, sulforaphane or a derivative thereof, and a broccoli extract. The present invention provides compositions related to these combinations.

[0031] The present invention also provides methods comprising administering these combinations. In some embodiments, the combinations can be administered to a subject to treat, prevent, reduce the occurrence of, reduce symptoms associated with, and / or reduce secondary recurrence of cancer, particularly breast cancer, prostate cancer, colon cancer, lung cancer, and bladder cancer, including administering to the subject. In some embodiments, the combinations can be administered to increase the level or gene expression of NAD(P)H:quinone oxidoreductase 1 (NQO-1) in a subject. In some embodiments, the combinations can be administered to treat, prevent, reduce the occurrence of, reduce symptoms associated with, and / or reduce secondary recurrence of diseases or conditions associated with elevated levels of quinone estrogens.

[0032] Sulforaphane is also known as 1-isothiocyanato-4-methylsulfinylbutane. Derivatives of sulforaphane include, but are not limited to, the sulfoxythiocarbamate analog of sulforaphane, 6-methylsulfinylhexyl isothiocyanate (6-HITC), and compounds containing the sulforaphane structure with different side chains and / or spacers of various lengths between the isothiocyanato and sulfoxide groups. Examples of sulforaphane derivatives include those described in the following references, each of which is incorporated by reference in its entirety: Hu et al., Eur J Med Chem, 2013, 64:529-539; Ahn et al., Proc Natl Acad Sci USA, 2010, 107(21):9590-9595; and Morimistu et al., J. Biol. Chem. 2002, 277:3456-3463; and Baird et al., Arch Toxicol, 2011, 85(4):241-272.

[0033] In some embodiments, the composition contains sulforaphane or a derivative thereof, preferably sulforaphane, in an amount of about 1 μg to about 10 g, preferably about 3 μg to about 5 g, preferably about 5 μg to about 1000 mg, preferably about 7 μg to about 750 mg, more preferably about 10 μg to about 500 mg, and most preferably about 100 μg to about 100 mg. In some embodiments, a composition suitable for human use contains about 1 mg to about 20 mg.

[0034] In some embodiments, the methods of the present invention involve administering to a subject sulforaphane or a derivative thereof, preferably sulforaphane, in an amount of about 1 μg to about 10 g, preferably about 3 μg to about 5 g, preferably about 5 μg to about 1000 mg, preferably about 7 μg to about 750 mg, more preferably about 10 μg to about 500 mg, and most preferably about 100 μg to about 100 mg. In some embodiments, where the subject is a human, the methods involve administering about 1 mg to about 20 mg. In some embodiments, the methods of the present invention involve administering to a subject sulforaphane or a derivative thereof, preferably sulforaphane, in an amount of about 0.01 μg / kg to about 0.2 g / kg, preferably about 0.05 μg / kg to about 0.07 g / kg, more preferably about 0.07 μg / kg to about 15 mg / kg, more preferably about 0.1 μg / kg to about 11 mg / kg, and most preferably about 0.2 μg / kg to about 7 mg / kg. In some embodiments, in which the subject is a human, the method comprises administering about 2 μg / kg to about 2 mg / kg, more preferably about 0.01 mg / kg to about 0.3 mg / kg. The above amounts may refer to the administration of individual doses or to the total daily dose. The total daily dose refers to the total amount of compound or component administered to a subject in a 24-hour period.

[0035] In some embodiments, the method includes administering two or more of sulforaphane or its derivatives. In some embodiments, the composition includes two or more of sulforaphane or its derivatives. For example, the method or composition may include both sulforaphane and one or more of its derivatives, or two or more of its derivatives. In some embodiments, the method or composition includes two or more of sulforaphane or its derivatives, the amount may refer to the amount of each sulforaphane or its derivative or the total amount of two or more sulforaphanes or their derivatives.

[0036] The term "sulforaphane precursor" refers to any compound, substance, or material that can be used to produce sulforaphane. In preferred embodiments, sulforaphane precursors preferably include compounds that can be converted or metabolized to sulforaphane by enzymes. In some preferred embodiments, sulforaphane precursors include glucoraphanin. Glucoraphanin is a glucosinolate also known as 4-methylsulfinylbutyl glucosinolate and 1-S-[(1E)-5-(methylsulfinyl)-N-(sulfonatooxy)pentanimidoyl]-1-thio-BD-glucopyranose.

[0037] In some embodiments, the composition contains about 1 μg to about 10 g, preferably about 250 μg to about 5 g, more preferably about 500 μg to about 2000 mg, even more preferably about 1 mg to about 750 mg, even more preferably about 1.5 mg to about 250 mg, even more preferably about 2 mg to about 100 mg, and most preferably about 3 mg to about 75 mg of a sulforaphane precursor, preferably glucoraphanin. In some embodiments, a composition suitable for human use contains about 3.5 mg to about 50 mg of a sulforaphane precursor, preferably glucoraphanin.

[0038] In some embodiments, the method comprises administering to the subject about 1 μg to about 10 g, preferably about 250 μg to about 5 g, more preferably about 500 μg to about 2000 mg, even more preferably about 1 mg to about 750 mg, even more preferably about 1.5 mg to about 250 mg, even more preferably about 2 mg to about 100 mg, and most preferably about 3 mg to about 75 mg of a sulforaphane precursor, preferably glucoraphanin. In some embodiments, where the subject is a human, the method comprises administering about 3.5 mg to about 50 mg. In some embodiments, the method comprises administering to the subject an amount of sulforaphane precursor in an amount of about 1 μg / kg to about 1000 mg / kg, preferably about 5 μg / kg to about 500 mg / kg, more preferably about 7.5 μg / kg to about 100 mg / kg, even more preferably about 10 μg / kg to about 25 mg / kg, and most preferably about 25 μg / kg to about 10 mg / kg. In some embodiments, in which the subject is a human, the method comprises administering about 50 μg / kg to about 800 μg / kg. The above amounts may refer to the administration of individual doses or the total daily dose.

[0039] In some embodiments, the method includes administering two or more sulforaphane precursors. In some embodiments, the composition includes two or more sulforaphane precursors. In some embodiments, where the method or composition includes two or more sulforaphane precursors, the amounts above can refer to the amount of each sulforaphane precursor or the total amount of sulforaphane precursors.

[0040] Sulforaphane precursors can be converted or metabolized to sulforaphane. In some embodiments, sulforaphane precursors are converted to sulforaphane by enzymes. In some embodiments, the enzyme capable of converting sulforaphane precursors to sulforaphane comprises a glucosidase enzyme, preferably a thioglucosidase enzyme, more preferably a myrosinase. Myrosinase is also known as thioglucoside glucohydrolase.

[0041] In some embodiments, the composition contains the enzyme in an amount of about 1 pg to about 1 μg, preferably about 50 pg to about 500 ng, and most preferably about 1 ng to about 150 ng. In some embodiments, compositions suitable for human use contain about 5 ng to about 75 ng of the enzyme.

[0042] In some embodiments, the method comprises administering an enzyme, preferably myrosinase, in an amount of about 1 pg to about 1 μg, preferably about 50 pg to about 500 ng, and most preferably about 1 ng to about 150 ng. In some embodiments, where the subject is a human, the method comprises administering about 5 ng to about 75 ng of the enzyme. In some embodiments, the method comprises administering to the subject an amount of the enzyme of about 0.02 pg / kg to about 0.02 μg / kg, preferably about 0.7 pg / kg to about 7 ng / kg, and most preferably about 0.02 ng / kg to about 2 ng / kg. In some preferred embodiments, where the subject is a human, the method comprises administering about 0.1 ng / kg to about 1 ng / kg. The above amounts may refer to the administration of individual doses or the total daily dose.

[0043] In some embodiments, the methods include administering two or more enzymes capable of converting a sulforaphane precursor to sulforaphane. In some embodiments, the compositions include two or more enzymes capable of converting a sulforaphane precursor to sulforaphane. In some embodiments, where the methods or compositions include two or more enzymes, the amounts above can refer to the amount of each enzyme or the total amount of enzymes.

[0044] The present invention also provides uses of broccoli extracts and / or powders, including, but not limited to, broccoli seed and sprout extracts and powders. The present invention also provides methods of administering the broccoli extracts and / or powders and compositions comprising the broccoli extracts and / or powders. In some embodiments, the broccoli extracts or powders are standardized to contain about 1% to about 75% by weight of a sulforaphane precursor, preferably glucoraphanin, more preferably about 2.5% to about 50%, even more preferably about 5% to about 25%, and most preferably about 10% to about 20%. Examples of broccoli extracts and powders include, but are not limited to, those described in U.S. Patent Nos. 5,411,986; 5,725,895; 5,968,505; 5,968,567; 6,177,122; 6,242,018; 6,521,818; 7,303,770 and 8,124,135, each of which is incorporated by reference in its entirety. Broccoli powder can be obtained, for example, by air-drying, freeze-drying, drum-drying, spray-drying, heat-drying and / or partial vacuum-drying broccoli, preferably broccoli sprouts. In some embodiments, compositions and methods include the use of about 1 μg to about 10 g, more preferably about 250 μg to about 5 g, even more preferably about 500 μg to about 1 g, preferably about 600 μg to about 500 mg, more preferably about 750 μg to about 400 mg, and most preferably about 1 mg to about 300 mg of broccoli extract. In some embodiments, the broccoli extract or powder is present in the composition or administered to the subject in an amount sufficient to provide the above amounts of sulforaphane precursor or sulforaphane. In some embodiments, the composition may further include an enzyme enhancer, preferably ascorbic acid. In some embodiments, the method may further include administration of an enzyme enhancer, preferably ascorbic acid.

[0045] Sulforaphane or its derivatives, sulforaphane precursors, and / or enzymes capable of converting sulforaphane precursors to sulforaphane can be obtained from any source, including, but not limited to, one or more plants from the Brassicaceae family (also known as Cruciferae). Examples of plants from the Brassicaceae family include, but are not limited to, broccoli, Brussels sprouts, cauliflower, cabbage, horseradish, parsnip, radish, wasabi, watercress, and white mustard. In some preferred embodiments, the sulforaphane precursor, preferably glucoraphanin, and the enzyme, preferably myrosinase, are obtained from broccoli, broccoli sprouts, or broccoli seeds. The sulforaphane precursor and the enzyme may be obtained from the same or different sources. In some embodiments, both the sulforaphane precursor and the enzyme may be obtained from extracts or powders derived from these plants, preferably broccoli seed or sprout extracts or powders.

[0046] The present invention provides the use of enzyme enhancers. The enzyme enhancers can be used to enhance the activity of enzymes capable of converting sulforaphane precursors to sulforaphane. In some embodiments, the enzyme enhancers include an enzyme cofactor, preferably ascorbic acid. Ascorbic acid, also known as ascorbate or vitamin C, can enhance the activity of myrosinase. In some embodiments, without an enzyme enhancer such as ascorbic acid, the conversion reaction to sulforaphane may be too slow and not occur at the site required for peak absorption. The enzyme enhancer may be obtained from natural sources or synthetically produced.

[0047] In some embodiments, the composition may contain from about 1 mg to about 500 mg, preferably from about 1 mg to about 250 mg, and most preferably from about 1 mg to about 125 mg of the enzyme enhancer. In some embodiments, a composition suitable for human use contains from about 1 mg to about 50 mg of the enzyme enhancer.

[0048] In some embodiments, the methods of the present invention involve administration of an enzyme-enhancing substance, preferably ascorbic acid, in an amount of about 1 mg to about 500 mg, preferably 1 mg to about 250 mg, and most preferably about 1 mg to about 125 mg. In some embodiments, in which the subject is a human, the methods involve administration of about 1 mg to about 50 mg. In some embodiments, the methods of the present invention involve administration of an enzyme-enhancing substance, preferably ascorbic acid, in an amount of about 0.01 mg / kg to about 3 mg / kg, most preferably about 0.02 mg / kg to about 2 mg / kg. In some embodiments, in which the subject is a human, the methods involve administration of about 0.02 mg / kg to about 0.7 mg / kg of the enzyme-enhancing substance. The amounts above may refer to the administration of individual doses or the total daily dose.

[0049] In some embodiments, the methods include administration of two or more enzyme-enhancing substances. In some embodiments, the compositions include two or more enzyme-enhancing substances. In some embodiments, where the methods or compositions include two or more enzyme-enhancing substances, the amounts above can refer to the amount of each enzyme-enhancing substance or the total amount of enzyme-enhancing substances.

[0050] The present invention provides the use of mushroom extracts or powders. In some embodiments, the mushrooms may include "medicinal mushrooms," including, but not limited to, maitake, shiitake, reishi, cremini, almond, chestnut, wood ear, cloud ear, porcini, ink cap, yarta gunbu, enokitake, shimeji, tiger milk, morel, bamboo, tamogitake, pink oyster, king oyster mushroom, oyster mushroom, cauliflower, white wood ear, golden jelly, matsutake, Mexican truffle, and straw mushroom. In a preferred embodiment, the mushrooms include maitake, shiitake, reishi, and / or a mixture of one or more of these.

[0051] Maitake mushrooms belong to the Grifola frondosa species. Maitake mushrooms may contain several fractions with biological activity. Examples of components found in Maitake mushrooms include, but are not limited to: glucans (such as α-glucans and β-glucans); lipids (such as octadecanoic acid and octadecadienoic acid); phospholipids (such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and phosphatidic acid).

[0052] Shiitake mushroom belongs to the Lentinula edodes species. Shiitake mushroom may contain some fractions with biological activity. Examples of components found in Shiitake mushroom include, but are not limited to, glucans (such as α-glucans and β-glucans), proteins (such as lentin); lipids (such as linoleic acid); and lignin.

[0053] Reishi mushrooms belong to the Ganoderma lucidum species. Reishi mushrooms may contain several fractions with biological activity. Examples of components found in Reishi mushrooms include, but are not limited to: glucans (α-glucan and β-glucan), canthaxanthin, sterols, coumarin, ganoderic acid, and mannitol.

[0054] In some preferred embodiments, the mushroom extract or powder contains one or more glucans. Glucans are polysaccharides of D-glucose monomers linked by glycosidic bonds and can be of the α- or β-type. In some embodiments, the glucans include one or more α-glucans and / or β-glucans. α-glucans include, but are not limited to, 1,4-α-glucans and 1,6-α-glucans, and β-glucans include, but are not limited to, 1,3-β-glucans, 1,4-β-glucans, and 1,6-β-glucans. Glucans can be expressed in various polymer configurations. In a preferred embodiment, the Maitake mushroom extract or powder contains 1,3-β-glucans and / or 1,6-β-glucans. In a preferred embodiment, the Shiitake mushroom extract or powder contains 1,4-α-glucans. In a preferred embodiment, the Reishi mushroom extract or powder contains 1,3-β-glucans and / or 1,6-β-glucans. In some embodiments, the compositions and methods of the present invention may involve the use of purified forms of glucan or synthetically produced glucan in place of mushroom extracts or powders.

[0055] In some embodiments, a maitake mushroom extract or powder may be used. In some embodiments, the maitake mushroom extract or powder is standardized to contain about 1% to about 75%, more preferably about 5% to about 50%, even more preferably about 10% to about 30%, and most preferably about 15% to about 20% of one or more glucans, preferably β-glucans, more preferably 1,3-β-glucans and / or 1,6-β-glucans. Examples of maitake mushroom extracts and powders include, but are not limited to, those described in U.S. Patent No. 5,854,404; WO2007142130; EP0893449; WO2009063885; WO2006107208; WO2007024496; and WO2001054673, each of which is incorporated by reference in its entirety. Maitake mushroom powder can be obtained, for example, by air-drying, freeze-drying, drum-drying, spray-drying, heat-drying, and / or partial vacuum-drying Maitake mushrooms. In some embodiments, the composition contains about 250 μg to about 100 mg, preferably about 500 μg to about 75 mg, and most preferably about 750 μg to about 50 mg. In some embodiments, a composition suitable for humans contains about 1 mg to about 20 mg of Maitake mushroom extract. In some embodiments, the method involves administering about 250 μg to about 100 mg, preferably about 500 μg to about 75 mg, and most preferably about 750 μg to about 50 mg. In some embodiments, in which the subject is a human, the method involves administering about 1 mg to about 20 mg of Maitake mushroom extract. The amounts above may refer to the administration of individual doses or the total daily dose.

[0056] In some embodiments, a shiitake mushroom extract or powder may be used. In some embodiments, the shiitake mushroom extract or powder is standardized to contain about 1% to about 75%, preferably about 10% to about 60%, even more preferably about 25% to about 50%, and most preferably about 30% to about 40% of one or more glucans, preferably α-glucans, more preferably 1,4-α-glucans. Examples of shiitake mushroom extracts include, but are not limited to, those described in U.S. Patent Nos. 5,780,097; 6,582,723; WO2005107496; WO2007024496; and WO2000033069, each of which is incorporated by reference in its entirety. Maitake mushroom powder may be obtained, for example, by air-drying, freeze-drying, drum-drying, spray-drying, heat-drying, and / or partial vacuum-drying maitake mushrooms. In some embodiments, the composition comprises about 1 mg to about 1 g, preferably about 10 mg to about 500 mg, and most preferably about 25 mg to about 300 mg. In some embodiments, a composition suitable for humans comprises about 50 mg to about 250 mg of shiitake mushroom extract or powder. In some embodiments, the method comprises administering about 1 mg to about 1 g, preferably about 10 mg to about 500 mg, and most preferably about 25 mg to about 300 mg. In some preferred embodiments, in which the subject is a human, the method comprises administering about 50 mg to about 250 mg of shiitake mushroom extract or powder to the subject. The amounts above may refer to individual doses or total daily doses. The amounts above may refer to individual doses or total daily doses.

[0057] In some embodiments, a Reishi mushroom extract or powder may be used. In some embodiments, the Reishi mushroom extract contains about 1% to about 75%, more preferably about 5% to about 50%, even more preferably about 10% to about 30%, and most preferably about 15% to about 20% of one or more glucans, preferably β-glucans, more preferably 1,3-β-glucans and / or 1,6-β-glucans. Maitake mushroom powder may be obtained, for example, by air-drying, freeze-drying, drum-drying, spray-drying, heat-drying, and / or partial vacuum-drying Maitake mushrooms.

[0058] In some embodiments, the compositions and / or methods include the use of one mushroom extract or powder, such as a Maitake mushroom extract or powder, a Shiitake mushroom extract or powder, or a Reishi mushroom extract or powder. In some embodiments, the compositions and / or methods include the use of a mixture of one or more types of mushroom extract or powder. In some embodiments, the compositions and / or methods include the use of a mixture of one or more of a Maitake mushroom extract or powder, a Shiitake mushroom extract or powder, and a Reishi mushroom extract or powder. The compositions and methods may include the use of an extract or powder or a mixture of an extract and a powder.

[0059] The present invention also provides for the use of any glucan-rich ingredient in place of or in addition to mushroom extract or powder. An example of a glucan-rich ingredient is baker's yeast. In some embodiments, a yeast preparation may be used. In some embodiments, the yeast preparation contains about 0.1% to about 50%, preferably about 0.5% to about 25%, and most preferably about 0.5% to about 10% of one or more glucans. Examples of yeast preparations include those discussed in U.S. Patent No. 5,223,491 and U.S. Patent No. 5,576,015, each of which is incorporated by reference in its entirety.

[0060] The method of the present invention can further comprise the administration of one or more additional components.The composition of the present invention can further comprise one or more additional components.The additional components can include active pharmaceutical ingredients, dietary supplements and nutritional extracts.Examples of additional components include, but are not limited to, ursolic acid, quercetin or its derivatives, amino sugars such as glucosamine, glycosaminoglycans such as chondroitin, avocado / soybean unsaponifiables, vitamins such as vitamin K2, coffee fruit, magnesium, ursolic acid, proanthocyanidins, α- and β-glucans, curcumin, phytosterols, phytostanols and S-adenosylmethionine (SAMe). These additional components may be present in milk thistle (Silybum marianum) extract (silymarin), cranberry (Vaccinium macrocarpon) extract (proanthocyanidins, quercetin, and ursolic acid), and turmeric (Curcuma longa).

[0061] In some embodiments, the ratio of β-glucan to sulforaphane or its derivative (β-glucan:sulforaphane or its derivative) is about 50:1 to about 1:50, preferably about 25:1 to about 1:25, more preferably about 10:1 to about 1:20, more preferably about 5:1 to about 1:10, even more preferably about 1:1 to about 1:8, and most preferably about 1:3 to about 1:5. In some embodiments, the ratio of α-glucan to sulforaphane or its derivative (α-glucan:sulforaphane or its derivative) is about 1:50 to about 50:1, preferably about 1:10 to about 25:1, more preferably about 1:5 to about 20:1, more preferably about 1:1 to about 15:1, even more preferably about 2:1 to about 10:1, and most preferably about 3:1 to about 8:1. In some embodiments, the ratio of β-glucan to sulforaphane precursor (β-glucan:sulforaphane precursor) is about 50:1 to about 1:50, preferably about 30:1 to about 1:35, more preferably about 20:1 to about 1:25, more preferably about 10:1 to about 1:20, even more preferably about 5:1 to about 1:15, and most preferably about 1:1 to about 1:10. In some embodiments, the ratio of α-glucan to precursor (α-glucan:precursor) is about 1:50 to about 100:1, preferably about 1:25 to about 75:1, more preferably about 1:10 to about 50:1, more preferably about 1:5 to about 40:1, even more preferably about 1:1 to about 30:1, and most preferably about 2:1 to about 20:1.

[0062] In some embodiments, the composition comprises a unit dosage form, including but not limited to, pharmaceutical dosage forms suitable for oral, rectal, intravenous, subcutaneous, intramuscular, transdermal, transmucosal, and topical administration. In some preferred embodiments, the composition comprises an orally administrable dosage form or a rectally administrable dosage form. Examples of orally administrable dosage forms include, but are not limited to, tablets, capsules, powders dispersible in beverages, liquids such as solutions, suspensions, or emulsions, soft gels / chewable capsules, chewable bars, or other convenient dosage forms known in the art. In preferred embodiments, the composition comprises a tablet, capsule, or soft chewable treat. Orally administrable dosage forms can be formulated for immediate release, sustained release, or delayed release.

[0063] In some embodiments, at least the sulforaphane precursor, enzyme, and enzyme enhancer are provided in a dosage form that allows for release in a region of the gastrointestinal tract having a pH of at least 4, preferably at least 5, e.g., the small intestine, preferably the duodenum. In some embodiments, at least sulforaphane or a derivative thereof and / or broccoli extract or powder are provided in a dosage form that allows for release in a region of the gastrointestinal tract having a pH of at least 4, preferably at least 5, e.g., the small intestine, preferably the duodenum. In some embodiments, the mushroom extract or powder and / or any optional additional components are also released in a region of the gastrointestinal tract having a pH of at least 4, preferably at least 5, e.g., the small intestine, preferably the duodenum. The small intestine includes the duodenum, jejunum, and ileum.

[0064] In some embodiments, each of these components (i.e., sulforaphane precursor, enzyme, enzyme enhancer, sulforaphane or a derivative thereof, broccoli extract or powder, mushroom extract or powder, and / or additional components) is released simultaneously or concomitantly (i.e., within a short period of each other). This provides an advantage over glucoraphanin-containing compositions formulated to release glucoraphanin in regions of the gastrointestinal tract having a pH below 4, e.g., the stomach. In such low pH environments, the acidic environment may result in conversion of the sulforaphane precursor to other physiologically inactive end-products, e.g., sulforaphane nitrile and epithionitrile.

[0065] In some embodiments, the composition may comprise an orally administrable composition comprising an enteric-coated dosage form or any dosage form that is resistant to degradation in regions of the gastrointestinal tract with a pH of less than 4, such as the stomach. For example, the orally administrable composition may comprise a tablet or capsule containing an enteric coating. The enteric coating may comprise materials including, but not limited to, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, polyvinyl acetate phthalate, methacrylic acid copolymer, methacrylic acid:acrylic acid ester copolymer, hydroxypropylmethylcellulose acetate succinate, hydroxypropylmethylcellulose trimellitate, shellac, cellulose acetate trimellitate, carboxymethylethylcellulose, and mixtures thereof. The enteric coating may comprise any suitable enteric polymer known in the art. In some embodiments, one or more of the components in the composition may be embedded in a matrix of an enteric polymer. In some embodiments, the orally administrable composition comprises a capsule that dissolves slowly in stomach acid and travels to the small intestine, such as DRCAPS™ acid-resistant capsules marketed by CAPSUGEL® or any other acid-resistant capsule.

[0066] In the most preferred form, the orally administrable composition is surrounded by a coating that will not dissolve unless the surrounding medium has a pH of at least 4, more preferably at least 5. Alternatively, a coating that controls release by time rather than pH can be used, with the rate adjusted so that the ingredient is not released until after the pH of the gastrointestinal tract has risen to at least 4, more preferably at least 5. Thus, sustained-release formulations can be used to prevent the presence of sulforaphane precursors, enzymes and enzyme enhancers capable of converting sulforaphane precursors to sulforaphane, or sulforaphane itself in the stomach. Using standard coating techniques, a coating layer(s) can be applied to the orally administrable composition. Enteric coating materials can be dissolved or dispersed in organic or aqueous solvents. The pH at which the enteric coat dissolves can be controlled by the selected polymer or polymer combination and / or the ratio of pendant groups. For example, the dissolution characteristics of a polymer film can be altered by the ratio of free carboxyl groups to ester groups. The enteric coating layer also contains a pharmaceutically acceptable plasticizer such as triethyl citrate, dibutyl phthalate, triacetin, polyethylene glycol, polysorbate, or other plasticizer. Additives such as dispersants, colorants, anti-adherents, and anti-foaming agents may also be included.

[0067] The composition may contain one or more non-active pharmaceutical ingredients (also commonly known as "excipients"). Non-active ingredients serve, for example, to solubilize, suspend, thicken, dilute, emulsify, stabilize, preserve, protect, color, flavor, and shape the active ingredient into a safe, convenient, otherwise acceptable, applicable, and effective preparation for use. Preferably, the excipient is a pharmaceutically acceptable excipient. Examples of classes of pharmaceutically acceptable excipients include lubricants, buffers, stabilizers, foaming agents, dyes, colorants, flavorings, fillers, bulking agents, fragrances, release-modifying agents, adjuvants, plasticizers, glidants, mold release agents, polyols, granulating agents, diluents, binders, buffers, absorbents, glidants, adhesives, anti-adherents, acidulants, emollients, resins, demulcents, solvents, surfactants, emulsifiers, elastomers, and mixtures thereof.

[0068] In some embodiments, the combination of (i) a sulforaphane precursor, preferably glucoraphanin, (ii) an enzyme capable of converting the sulforaphane precursor to sulforaphane, preferably a glucosidase enzyme, more preferably a thioglucosidase enzyme, and most preferably myrosinase, (iii) an enzyme enhancer, preferably an enzyme cofactor, more preferably ascorbic acid, and (iv) a mushroom extract or powder (containing glucan) exhibits synergistic effects. In some embodiments, the combination of sulforaphane (or a derivative thereof) and a mushroom extract or powder (containing glucan) exhibits a synergistic effect. Synergy refers to the effect of a combination of two or more ingredients providing a result that is greater than the sum of the effects provided by the substances when used alone. In preferred embodiments, the synergistic effect is greater than an additive effect. In some embodiments, the combination of a sulforaphane precursor, an enzyme capable of converting the sulforaphane precursor to sulforaphane, an enzyme enhancer, and a Maitake, Shiitake, or Reishi mushroom extract or powder has a statistically significantly greater effect than (i) each component alone, (ii) the combination of the sulforaphane precursor and enzyme alone, and / or (iii) the combination of the sulforaphane precursor, enzyme, and enzyme enhancer alone.

[0069] In preferred embodiments, the combination of sulforaphane precursor, enzyme, enzyme enhancer, and mushroom extract or powder (containing glucan) exhibits synergy by having a statistically significant and / or greater than additive effect compared to the sulforaphane precursor alone and the mushroom extract or powder alone. In some embodiments, the combination of glucoraphanin, myrosinase, ascorbic acid, and mushroom extract or powder has a synergistic effect compared to the combination of glucoraphanin, myrosinase, and ascorbic acid alone, and compared to glucan alone.

[0070] In some embodiments, the combination of sulforaphane (or a derivative thereof) and mushroom extract or powder has a statistically significant and / or greater than additive effect than (i) sulforaphane (or a derivative thereof) alone and / or (ii) mushroom extract or powder alone. In some embodiments, the combination of sulforaphane and glucan has a synergistic effect compared to sulforaphane alone and glucan alone.

[0071] In some embodiments, the combination of broccoli extract or powder and mushroom extract or powder has a statistically significant and / or greater than additive effect than (i) broccoli extract or powder alone and / or (ii) mushroom extract or powder alone. In some embodiments, the combination of broccoli extract or powder and glucan has a synergistic effect compared to broccoli extract or powder alone and glucan alone.

[0072] The present invention provides methods of use, including administration to a subject in need thereof. In some embodiments, the methods include administering a combination of a sulforaphane precursor, an enzyme capable of converting the sulforaphane precursor to sulforaphane, an enzyme enhancer, and a mushroom extract or powder. In some embodiments, the methods include administering a combination of sulforaphane or a derivative thereof and a mushroom extract or powder. In some embodiments, the methods include administering a combination of broccoli extract or powder and a mushroom extract or powder.

[0073] In some embodiments, the methods relate to treating, preventing, reducing the incidence of, reducing symptoms associated with, and / or reducing secondary recurrence of cancer in a subject, particularly breast cancer, prostate cancer, colon cancer, lung cancer, liver cancer, and bladder cancer. The methods may be useful in reducing or slowing damage to tissues and organs such as the breast, prostate, colon, lung, liver, and bladder. The present invention provides methods for treating, preventing, reducing symptoms associated with, and / or reducing secondary recurrence of diseases and conditions associated with the reproductive system (including, but not limited to, the breast and prostate), colon, liver, bladder, kidney, central nervous system, cardiovascular system, pulmonary system, genitourinary system, hematopoietic system, and joints. The present invention also provides methods for treating, preventing, reducing symptoms associated with, and / or reducing secondary recurrence of cysts, such as benign cysts.

[0074] In some embodiments, the method relates to increasing the level of NAD(P)H:quinone oxidoreductase 1 (NQO-1) or increasing its gene expression in a subject.The method can also be useful in treating, preventing, reducing the symptoms associated with, and / or reducing the secondary recurrence of diseases and conditions that will benefit from increasing the gene expression or level of NQO-1.Examples of such diseases and conditions include, but are not limited to, cancer, myelodysplastic syndrome, cardiovascular disease, and tardive dyskinesia.

[0075] In some embodiments, the methods relate to treating, preventing, reducing the occurrence, diminishing symptoms associated therewith, and / or reducing secondary recurrence of diseases or conditions associated with elevated levels of quinone estrogens, including, but not limited to, cancer, myelodysplastic syndrome, cardiovascular disease, and tardive dyskinesia.

[0076] In some embodiments, the methods relate to providing beneficial effects on biomarkers and treating, preventing, reducing the occurrence of, or diminishing symptoms associated with abnormal levels of these biomarkers. Examples of such biomarkers include, but are not limited to, NADPH-dependent enzymes, thioredoxin (TXN), thioredoxin reductase-1 (Txnrd-1), glutamate-cysteine ​​ligase subunit (GCLC), sulfotransferase 1A1 (SULT1A1), heme oxygenase-1 (HMOX1), glutathione peroxidase-3 (GPx-3), glutathione S-transferase theta 2 (GSTT2), microsomal glutathione S-transferase 1 (MG), and the like. These include ST1), aldehyde oxidase (AOX1), aldo-keto reductase 1B8 (Akr1b8), flavin-containing monooxygenase 2 (FMO2), Fe receptor domain receptor III (Fcgr3), tryptase beta 1 (TPSB1), mast cell protease-6 (Mcpt6), neurexin-1-alpha (NRXN-1), microphthalmia-associated transcription factor (MITF), type II iodothyronine deiodinase (DIO2), angiopoietin-14 (Angpt14), differentiation antigen (CD36), and Ntel. Diseases or conditions associated with elevated or abnormal levels of these biomarkers include, but are not limited to, cancer, pulmonary and central nervous system tuberculosis, multiple sclerosis, Crohn's disease, atherosclerosis, osteoarthritis, asthma, stroke, emphysema, diabetic nephropathy, chronic histiocytic placental villositis, hypertension, abdominal aortic aneurysm, inflammatory bowel disease, chronic sinusitis, coronary artery disease, and kidney disease.

[0077] In some embodiments, the method comprises administering to a subject in need thereof a combination of a mushroom extract or powder containing sulforaphane and glucan. In some embodiments, the method comprises administering to a subject in need thereof a combination of a broccoli extract or powder and a mushroom extract or powder containing glucan. In some preferred embodiments, the method comprises administering to a subject a combination of a mushroom extract or powder containing glucoraphanin, myrosinase, ascorbic acid, and glucan. In preferred embodiments, the combination exhibits a synergistic effect in the methods of the present invention.

[0078] In preferred embodiments, one or more components of the combination (e.g., sulforaphane precursor, enzyme capable of converting sulforaphane precursor to sulforaphane, enzyme enhancer, mushroom extract or powder; or sulforaphane or a derivative thereof and mushroom extract or powder; or broccoli extract or powder and mushroom extract or powder) are administered together in a single composition or dosage form or separately, preferably within a period of overlapping therapeutic properties. In some embodiments, the components of the combination may be administered in two or more orally administrable compositions or dosage forms. For example, in some embodiments, the sulforaphane precursor, enzyme capable of converting sulforaphane precursor to sulforaphane, and enzyme enhancer are administered in a single orally administrable dosage form, while the mushroom extract or powder is administered in one or more separate or additional orally administrable dosage forms. In preferred embodiments, the components of the combination are administered in a single dosage form.

[0079] In some embodiments, the combination may be administered 1 to 10 times per day, preferably 1 to 5 times per day, more preferably 1 to 3 times per day, and most preferably once per day.

[0080] The dosages disclosed herein preferably refer to dosages suitable for humans. Dose calculations can be determined by those skilled in the art by assessing body weight, surface area, metabolic rate and species differences.

[0081] The term "subject" refers to any animal, including mammals and birds. Mammals include, but are not limited to, humans, dogs, cats, horses, cows, camels, elephants, lions, tigers, bears, sea lions, and rabbits. In a preferred embodiment, the subject includes mammals that are not consumed as food, such as humans, cats, and dogs. [Example]

[0082] formulation

[0083] The following are exemplary formulations of the present invention.

[0084] Formulation A Broccoli seed extract containing glucoraphanin (approximately 12% by weight), 50 mg to 5 grams, freeze-dried broccoli sprout powder containing myrosinase, 25 mg to 500 mg, ascorbic acid, 1 mg to 50 mg, shiitake mushroom extract containing alpha-glucan (approximately 40% by weight), 1 mg to 250 mg

[0085] Formulation B Broccoli seed extract containing glucoraphanin (approximately 12% by weight), 50 mg to 5 grams, freeze-dried broccoli sprout powder containing myrosinase, 25 mg to 500 mg, ascorbic acid, 1 mg to 50 mg, maitake mushroom extract containing beta-glucan (approximately 20% by weight), 1 to 100 mg

[0086] Formulation C An orally administrable composition comprising: Broccoli Seed Extract Broccoli sprout extract Maitake mushroom extract Ascorbic acid Hydroxypropyl methylcellulose Microcrystalline cellulose cornstarch Ethyl cellulose Croscarmellose sodium Sodium starch glycolate Crospovidone silicon dioxide Sodium Alginate Medium-chain triglycerides Maltodextrin oleic acid Magnesium stearate stearic acid [Example]

[0087] A hydrophobic interaction chromatography (HILIC) method was developed, including the following conditions: Column: Waters BEH Amide, 1.7 μm particle size; 2.1 mm x 100 mm Mobile phase: 20% 10 mM ammonium acetate, pH 5.0; 80% acetonitrile; Separation mode: Isocratic Column temperature: 70℃ Flow rate: 0.7ml / min The above conditions allow for the isolation of five typical cruciferous glucosinolates, including the sulforaphane precursor, glucoraphanin. [Example]

[0088] Glucoraphanin consumption as a function of ascorbic acid concentration Approximately 250 mg of broccoli seed extract containing approximately 12% glucoraphanin by weight was subjected to hydrolysis with a fixed concentration of broccoli sprout-derived myrosinase in the presence of variable concentrations of ascorbic acid ranging from 0 to 600 μmol / L. The reaction mixture was temperature-controlled at 38°C, and aliquots were removed every 15 minutes for 60 minutes, and the glucoraphanin concentration was determined by chromatography. The rate of glucoraphanin consumption was interpreted as its rate of conversion to sulforaphane. Graphical representation of the reduction in glucoraphanin content as a function of increasing ascorbic acid concentration resulted in a series of linear plots, with the slope of the linear regression line reflecting the rate of glucoraphanin consumption in μmol / min. It is clear that in the presence of ascorbic acid at a concentration of 600 μmol / L, the reaction rate increased 13-fold compared to that proceeding in the absence of the modulating effect of ascorbic acid.

[0089] [Table 1] [Example]

[0090] Equimolar conversion of glucoraphanin to sulforaphane

[0091] To further elucidate the role of ascorbic acid in regulating myrosinase activity, a two-part experiment was conducted. All solutions were prepared in 20 mM Tris-buffered saline at pH 7.5, previously identified as optimal for myrosinase activity; each sample tube contained an accurately weighed 100 mg of freeze-dried broccoli powder as the source of myrosinase. The experiment was carried out at 38°C for 2 hours, with sample aliquots withdrawn at 30-minute intervals and assessed for both glucoraphanin and sulforaphane content by HPLC. A strongly acidic "stop" solution was utilized to immediately inhibit further myrosinase activity in the withdrawn aliquots. Control samples contained no ascorbic acid, and enzymatic conversion proceeded without cofactor assistance.

[0092] Part 1. Increasing amounts of broccoli seed extract (approximately 12% glucoraphanin by weight) ranging from 250 mg to 500 mg were added in the presence of a fixed concentration of ascorbic acid (1 mmol / liter).

[0093] Part 2. Keeping the amount of broccoli seed extract fixed at 250 mg, the concentration of ascorbic acid was varied from 0.4 mmol / liter to 3.8 mmol / liter.

[0094] The following table shows glucoraphanin and sulforaphane expressed in μmoles. It is clear that the conversion of glucoraphanin to sulforaphane was complete within the first 30 minutes in most reaction mixtures. However, careful examination of the enzymatic conversion occurring in control samples without the stimulating effect of ascorbic acid indicates equimolar conversion of glucoraphanin to sulforaphane; i.e., the amount of glucoraphanin consumed results in an equal amount of sulforaphane produced.

[0095] [Table 2]

[0096] In part 2 of the experiment, the modulatory effect of increasing concentrations of ascorbic acid on myrosinase activity was evaluated. An initial, apparently linear increase in the myrosinase-promoted conversion of glucoraphanin to sulforaphane was observed up to an ascorbic acid concentration of approximately 2 mmol / L, followed by a significant leveling off thereafter.

[0097] Finally, examination of sulforaphane yield after 30 minutes in Part 1 of the experiment indicates that a fixed amount of myrosinase contained in 100 mg of freeze-dried broccoli sprout powder in the presence of 1 mmol / L ascorbic acid can produce at least 200 μmol of sulforaphane in a predictable, linear fashion. Figures 1, 2, 3, and 4 show the results of this study. [Example]

[0098] Conversion of glucoraphanin to sulforaphane in the presence of simulated intestinal fluid.

[0099] Simulated intestinal fluid (SIF) powder (a commercially available concentrate that closely approximates human intestinal contents in terms of composition, pH, and ionic strength) was used. Experiments were performed using a USP dissolution apparatus 2 (paddle type), in which 500 ml of simulated intestinal fluid was dispensed into six dissolution vessels along with 150 mg of freeze-dried broccoli sprout powder as a source of myrosinase. Ascorbic acid concentrations varied from 0.25 to 1.00 mmol / L in vessels 1–4; vessel 5 contained 1 mmol / L ascorbic acid plus 3.125 g of pancreatin (8x USP); vessel 6 contained 1 mmol / L ascorbic acid plus 3.125 g of pancreatin (8x USP) and double the amount of freeze-dried broccoli sprout powder (300 mg). After the vessels were brought to 38°C, 250 mg of glucoraphanin-rich (12% by weight) broccoli seed extract was added to each, and the resulting suspensions were stirred at 75 RPM for 2 hours. Aliquots were removed every 15 minutes and assayed for sulforaphane. Figure 4 shows a direct correlation between the high yield of sulforaphane and the high concentration of ascorbic acid, particularly in the first stage of the experiment. [Example]

[0100] The following study was conducted to examine the effects of a combination of sulforaphane and a 20% β-glucan-containing Maitake mushroom extract on the gene expression of Nad(P)H:quinone oxidoreductase 1 (NQO-1). NQO-1 encodes a protein that can metabolize estrogen quinones and prevent them from forming DNA adducts that cause mutations and ultimately carcinogenesis. Increased NQO-1 expression is beneficial for breast, colon, liver, lung, skin, and prostate health.

[0101] In this study, the macrophage cell line RAW264.7 was treated for 24 hours with DMSO (vehicle control), sulforaphane (SFN), Maitake mushroom extract (Grafica edodes) with approximately 20% β-glucan content, or a combination of sulforaphane and Maitake mushroom extract. Specifically, cells were treated with one of the following: (i) DMSO (vehicle control), (ii) 0.5 μM SFN, (iii) 250 μg / ml Maitake mushroom, (iv) 500 μg / ml Maitake mushroom, (v) 750 μg / ml Maitake mushroom, (vi) 0.5 μM SFN and 250 μg / ml Maitake mushroom, (vii) 0.5 μM SFN and 500 μg / ml Maitake mushroom, and (viii) 0.5 μM SFN and 750 μg / ml Maitake mushroom. Gene expression of NQO-1 was analyzed by quantitative RT-PCR. The results, presented in Figure 5, show that:

[0102] [Table 3]

[0103] The results show that the combination of sulforaphane and Maitake mushroom extract had a synergistic effect compared to each component alone. This effect was found to be more than simply additive. [Example]

[0104] The following study was conducted to investigate the effect of a combination of sulforaphane and a shiitake mushroom extract containing 40% alpha-glucan on the gene expression of Nad(P)H:quinonequinone oxidoreductase 1 (NQO-1).

[0105] In this study, the macrophage cell line RAW264.7 was treated for 24 hours with DMSO (vehicle control), sulforaphane (SFN), a shiitake mushroom extract (L. edodes) with at least 20% α-glucan content, or a combination of sulforaphane and L. edodes mushroom extract. Specifically, cells were treated with one of the following: (i) DMSO (vehicle control), (ii) 0.5 μM SFN, (iii) 100 μg / ml shiitake, (iv) 250 μg / ml shiitake, (v) 500 μg / ml shiitake, (vi) 0.5 μM SFN and 100 μg / ml shiitake, (vii) 0.5 μM SFN and 250 μg / ml shiitake, and (viii) 0.5 μM SFN and 500 μg / ml shiitake. Gene expression of NQO-1 gene expression was analyzed by quantitative RT-PCR. The results, presented in Figure 6, show the following:

[0106] [Table 4]

[0107] The results demonstrate that the combination of sulforaphane and shiitake mushroom extract had a synergistic effect compared to each component alone. This effect was found to be more than merely additive. [Example]

[0108] Subject presents with breast cancer and suffers from symptoms including damaged breast tissue and breast pain.This woman is administered with a tablet that contains glucoraphanin, myrosinase, ascorbic acid and Maitake mushroom extract.The tablet is an enteric coated formulation that releases its contents in the small intestine.After one month of daily administration of the tablet, subject experiences the adjustment of alternative biomarkers including NQO-1, which correlates with the improvement of symptoms. [Example]

[0109] The subject has breast cancer and suffers from symptoms including damaged breast tissue and breast pain.The subject is administered a tablet containing glucoraphanin, myrosinase, ascorbic acid and shiitake mushroom extract.The tablet is an enteric coated formulation that releases its contents in the small intestine.After one month of daily administration of the tablet, the subject shows NQO-, which correlates with symptom improvement. Experience modulation of surrogate biomarkers, including 1.

Claims

1. 1. A composition for increasing the level of or increasing gene expression of NAD(P)H:quinone oxidoreductase 1 (NQO-1) in a subject, comprising: The composition comprises a combination of sulforaphane and glucan as active ingredients, and the combination exhibits a synergistic effect between these active ingredients, The composition, wherein the glucan is provided as a Reishi mushroom extract or powder.

2. 10. The composition of claim 1, wherein the Reishi mushroom extract comprises from about 1 to about 75% by weight of one or more glucans.

3. The composition according to claim 2, wherein the one or more types of glucan are β-glucan and / or 1,3-β-glucan and / or 1,6-β-glucan.

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