RU2785683C2 - Compositions containing sulforaphane or sulforaphane precursor and extract or powder of milk thistle - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: group of inventions relates to the pharmaceutical industry, namely to a composition for oral administration for increasing the content of glutathione in the treatment of cancer selected from liver cancer, rectal cancer, and colon cancer. The composition for oral administration for increasing the content of glutathione in the treatment of cancer selected from liver cancer, rectal cancer, and colon cancer contains a combination of sulforaphane and an extract of milk thistle standardized to the content from 20 to 35 wt.% of silibinin. The composition for oral administration for increasing the content of glutathione in the treatment of cancer selected from liver cancer, rectal cancer, and colon cancer contains a combination of an extract or powder of broccoli standardized to the content from 10 to 20 wt.% of glucoraphanin and an extract of milk thistle standardized to the content from 20 to 35 wt.% of silibinin.

EFFECT: above-described compositions synergistically increase the content of glutathione in the treatment of cancer selected from liver cancer, rectal cancer, and colon cancer.

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Description

This application claims priority based on the following applications, each of which is incorporated herein by reference in its entirety: provisional application for US Patent No. 61/668328, filed July 5, 2012; provisional application for US Patent No. 61/668342, filed July 5, 2012; provisional application for US Patent No. 61/668386, filed July 5, 2012; provisional application for US Patent No. 61/668396, filed July 5, 2012; provisional application for US Patent No. 61/668364, filed July 5, 2012; provisional application for US Patent No. 61/668374, filed July 5, 2012 and provisional application for US Patent No. 61/794417, filed March 15, 2013.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to a combination of a sulforaphane precursor, an enzyme capable of converting a sulforaphane precursor to sulforaphane, an enzyme potentiator, and milk thistle extract or powder. The present invention also relates to the combination of sulforaphane or a derivative thereof and milk thistle extract or powder. The present invention also relates to a combination of broccoli extract or powder and milk thistle extract or powder. The present invention provides compositions and methods related to these combinations.

BACKGROUND OF THE INVENTION

The use of natural products is gaining increasing popularity among people and pets. Some of these natural products are included in dietary supplements and health food products. There is a need in the art for additives useful as chemoprotective agents and/or antioxidants. In addition, there is a need in the art for pharmaceutical compositions and dietary supplements useful in conditions and disorders associated with glutathione. Chemoprotection through the consumption of natural products is emerging as an effective, inexpensive, accessible, and practical means to prevent or reduce the incidence of many conditions that affect humans and pets. It is known that carcinogens capable of damaging cells at the molecular level are often ingested and inhaled as non-toxic precursors. These non-toxic precursors can subsequently be converted into carcinogens in the body. Chemoprotective agents, such as natural substances capable of activating detoxifying enzymes or their cofactors, can neutralize and remove carcinogens. These natural substances can enhance other natural defense mechanisms such as the immune system.

Some natural products have antioxidant activity. Oxidative stress plays a major role in aging, the development of neurodegenerative diseases, as well as in physiological trauma such as ischemia. Antioxidant agents may reduce or inhibit the oxidation of essential biomolecules and may play a role in the treatment, prevention, or reduction in the incidence of conditions caused by oxidative stress.

Some natural foods are good for liver health. Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the US, affecting about 30% of the population. NAFLD is also known as liver lipidosis. In 10% of subjects with NAFLD, the disease will progress to non-alcoholic steatohepatitis (NASH), of which 25% will develop cirrhosis. Potentially, 10-25% of these cirrhotic patients will develop hepatocellular carcinoma. With its current incidence and lack of effective treatments, hepatocellular carcinoma is expected to become the most commonly diagnosed cancer in the United States by 2030. NAFLD is caused by the accumulation of fat in the liver (steatosis), but not by excessive alcohol consumption.

NAFLD has been associated with chronic inflammation, insulin resistance, diabetes, and obesity. In NAFLD, there are no or mild symptoms, it is most often diagnosed by abnormal results obtained from routine blood tests (i.e., elevated serum ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels in the serum) and confirmed by ultrasound or biopsy. There are currently no surgical or pharmacological treatments for NAFLD. Recommendations for managing NAFLD include lifestyle changes such as eating healthy, exercising, losing weight, lowering cholesterol, and controlling diabetes.

NAFLD is often associated with a decrease in glutathione. Glutathione is a tripeptide with a gamma peptide bond between the amino group of cysteine and the carboxyl group of the glutamate side chain. Glutathione plays an important role in the body as an antioxidant, detoxifier, and immune booster. Glutathione can combine with metabolites and toxins such as pro-carcinogens to remove them from the body. Patients may experience low glutathione levels for a number of reasons, including malnutrition, pollution, exposure to toxins and/or certain drugs, stress, trauma, aging, infection, and radiation. Low levels of glutathione can make the patient susceptible to oxidative stress, increase morbidity and predisposition to cancer. For example, decreased glutathione levels have been associated with conditions related to the liver, prostate, brain, lungs, kidneys, colon, breast, esophagus, pancreas, ovaries, and so on. Examples of disorders associated with reduced and deficient glutathione include, but are not limited to: NAFLD, cancer (lung, prostate, colon, breast, brain, liver, ovary, esophagus, pancreas, nose and throat, osteosarcoma), leukemia, cystic fibrosis, HIV, glutathione synthetase deficiency, Alzheimer's disease, Parkinson's disease, Huntington's disease, Charcot's disease, Friedreich's hereditary ataxia, multiple sclerosis, fibromyalgia, chronic fatigue, autism and diabetes.

An example of a natural product believed to have chemoprotective and antioxidant properties is sulforaphane. Sulforaphane is an organic organosulfur compound, also known as 1-isothiocyanate-4-methylsulfinylbutane. The precursor of sulforaphane, glucoraphanin, can be obtained from vegetables in the Brassicaceae family such as broccoli, Brussels sprouts and cabbage. However, in order to obtain sufficient quantities from vegetables for chemoprotection, it is necessary to spend them in large volumes. Glucoraphanin is converted to sulforaphane by a thioglucosidase enzyme called myrosinase, available from a variety of exogenous resources such as Brassicaceae vegetables and endogenously from the gut microflora. However, after ingestion of glucoraphanin, not all animals are able to convert it to sulforaphane, most likely due to variations in microflora populations and general health. In addition, in an acidic environment, such as in the stomach, glucoraphanin can be converted to inert metabolites. The active metabolite, sulforaphane, excites nuclear erythroid-2-related factor (Nrf2), which in turn increases the production of phase II detoxifying enzymes and cytoprotective enzymes such as glutathione-S-transferase, NAD(P)H:quinone oxidoreductase (NQO1) and heme oxygenase-1 (HO-1). It is believed that sulforaphane stimulates the production of these enzymes, without significantly affecting the synthesis of p-450 cytochrome enzymes. Increased production of phase II enzymes is believed to play a role in a variety of biological processes, including protecting the brain from cytotoxicity, protecting the liver from the toxic effects of fat storage, and detoxifying many other tissues.

Sulforaphane and its precursor glucoraphanin have been extensively studied. Shapiro et al. (Nutrition and Cancer, (2006), Vol. 55(1), pp. 53-62) discusses a phase I clinical trial evaluating the safety, tolerability, and metabolism of glucosinolates and isothiocyanates from broccoli sprouts. Shapiro et al. discuss a randomized, double-blind, placebo-controlled clinical trial of sprout extracts containing either glucosinolates, such as glucoraphanin, or isocyanates, such as sulforaphane, in healthy subjects. The study showed that the administration of these substances did not lead to systemic, clinically significant side effects.

Milk thistle (Silybum marianum) is a plant of the Asteraceae family. Milk thistle contains silymarin, which is composed of a number of components including, but not limited to, flavonolignans such as silybinin (also known as silybin), isosilybinin, silycristin, silydianin, quercetin, dehydrosilybin, deoxysilicystin, deoxysilydianin, silyndrin, silybinome, silygermine, and neosilygermine. The components of silymarin may have a number of biological effects, including inhibition of free radical formation, scavenging of free radical species, prevention of lipid peroxidation, increase in glutathione, and iron chelation. Silibinin is the most active component of silymarin and is believed to have hepatoprotective properties. Silymarin is discussed in US Patent No. 756377 9, which is incorporated herein in its entirety by reference.

Zhang et al. (Proc. Natl. Acad. Sci., (1994), Vol. 91, pp. 3147-3150) describe a study in Sprague-Dawley rats to determine the anticarcinogenic activity of sulforaphane and its structural analogs, synthetic norbornyl isothiocyanates. The study showed that sulforaphane administration was effective in blocking breast tumor formation.

Cornblatt et al. (Carcinogenesis, (2007), Vol. 38(7): pp. 1485-14 90) describe a study in Sprague-Dawley rats to determine the effect of sulforaphane in breast chemoprotection. The study showed that oral administration of any sulforaphane resulted in a threefold increase in NAD(P)H:quinone oxidoreductase (NQ01) enzymatic activity and a fourfold increase in heme oxygenase-1 (HO-1) enzyme immunostaining in the breast epithelium.

Munday et al. (Cancer Res, (2008), Vol. 68(5): pp. 1593-1600) describe a study regarding the effect of a freeze-dried aqueous extract of broccoli sprouts on the development of bladder cancer in rats. The study showed that the administration of broccoli sprout extract led to a significant induction of glutathione-3-transferase and NAD(P)H:quinone oxidoreductase 1 in the bladder, enzymes with protective activity against oxidants and carcinogens.

Aghazadeh S et al. (Exp Toxicol Pathol. (2011) Sep; 63(6):569-74) describe the anti-apoptotic and anti-inflammatory effects of Silybum marianum in the treatment of experimental steatohepatitis. The study showed that administration of an extract of Silybum maranium to rats fed a methionine-choline-deficient (MCD) diet to induce non-alcoholic steatohepatitis resulted in an increase in AST and ALT activity along with an increase in glutathione compared to control rats fed only the MCD diet.

European Patent Application No. 2213280 discloses compositions containing glucosinolates such as glucoraphanin and myrosinase, the composition being enclosed or coated.

All sources cited in this document are incorporated herein in their entirety by reference.

DISCLOSURE OF THE INVENTION

The present invention provides a composition comprising: (i) a sulforaphane precursor, preferably glucoraphanin; (ii) an enzyme capable of converting a sulforaphane precursor to sulforaphane, preferably a glucosidase enzyme, more preferably a thioglucosidase enzyme, and most preferably myrosinase; (iii) an enzyme potentiator, preferably ascorbic acid; and (iv) milk thistle extract or powder. The present invention also provides a method for treating, preventing, reducing the incidence, reducing symptoms, and/or reducing recurrences of a disease or condition associated with the liver, prostate, brain, lungs, kidneys, colon, breast, esophagus, pancreas, or ovaries 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 potentiator, and (iv) milk thistle extract or powder. The present invention also provides a method for increasing glutathione in a subject in need thereof, comprising administering to the subject: (i) a sulforaphane precursor, (ii) an enzyme capable of converting the sulforaphane precursor to sulforaphane, (iii) an enzyme potentiator, and (iv) a milk extract or powder. thistle. The present invention also provides a method for treating, preventing, reducing the incidence, reducing symptoms and/or reducing recurrence of a disease, non-alcoholic fatty liver disease (NAFLD) and/or any other liver disorder, comprising administering to a subject: (i) a sulforaphane precursor, (ii) an enzyme capable of converting the sulforaphane precursor to sulforaphane, (iii) an enzyme potentiator, and (iv) milk thistle extract or powder.

The present invention provides a composition comprising: (i) sulforaphane or a derivative thereof, and (ii) milk thistle extract or powder. The present invention also provides a method for treating, preventing, reducing the incidence, reducing symptoms, and/or reducing recurrences of a disease or condition associated with the liver, prostate, brain, lungs, kidneys, colon, breast, esophagus, pancreas, or ovaries. in a subject, comprising administering to the subject: (i) sulforaphane or a derivative thereof, and (ii) milk thistle extract or powder. The present invention also provides a method for increasing glutathione in a subject in need thereof, comprising administering to the subject: (i) sulforaphane or a derivative thereof, and (ii) milk thistle extract or powder. The present invention also provides a method of treating, preventing, reducing the incidence, reducing symptoms, and/or reducing recurrences of non-alcoholic fatty liver disease (NAFLD), and/or any other liver disorder, comprising administering to a subject: (i) sulforaphane or a derivative thereof, and ( ii) milk thistle extract or powder.

The present invention provides a composition comprising: (i) broccoli extract or powder and (ii) milk thistle extract or powder. The present invention also provides a method for treating, preventing, reducing the incidence, reducing symptoms, and/or reducing recurrences of a disease or condition associated with the liver, prostate, brain, lungs, kidneys, colon, breast, esophagus, pancreas, or ovaries. in a subject, comprising administering to the subject: (i) broccoli extract or powder; and (ii) milk thistle extract or powder. The present invention also provides a method of increasing glutathione in a subject in need thereof, comprising administering to the subject: (i) broccoli extract or powder and (ii) milk thistle extract or powder. The present invention also provides a method for the treatment, prevention, reduction in frequency, reduction in associated symptoms, and/or reduction in recurrence of non-alcoholic fatty liver disease (NAFLD) and/or any other liver disorder, comprising administering to a subject: (i) broccoli extract or powder, and ( ii) milk thistle extract or powder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scheme describing the conversion of glucoraphanin at 38°C without ascorbic acid as described in Example 4.

FIG. 2 is a diagram describing the conversion over about 10 minutes at 38°C as a function of ascorbic acid concentration, as described in Example 4.

FIG. 3 is a graph describing the conversion to sulforaphane over 30 minutes at 38°C and 1 mM ascorbic acid as described in Example 4.

FIG. 4 is a diagram describing the conversion of glucoraphanin to an intestinal fluid simulating fluid as described in Example 5.

FIG. 5 is a diagram describing the results of the experiment described in Example 6.

FIG. 6 is a diagram describing the results of the experiment described in Example 6.

IMPLEMENTATION OF THE INVENTION

The present invention relates to a combination of a sulforaphane precursor, an enzyme capable of converting a sulforaphane precursor to sulforaphane, an enzyme potentiator, and milk thistle extract or powder. The present invention also relates to the combination of sulforaphane or a derivative thereof and milk thistle extract or powder. The present invention also relates to a combination of broccoli extract or powder and milk thistle extract or powder. The present invention also relates to the use of milk thistle extract or powder in admixture with one or more of the following: sulforaphane precursor, sulforaphane or a derivative thereof, and broccoli extract. The present invention provides compositions related to these combinations.

The present invention provides methods comprising administering these combinations. In some embodiments, the combination may be administered to a subject to treat, prevent, reduce the incidence, reduce symptoms, and/or reduce recurrence of a disease or condition associated with the liver, prostate, brain, lungs, kidneys, colon, breast, esophagus , pancreas or ovaries. In some embodiments, a combination may be administered to a subject in need of treatment to increase the subject's glutathione content. In some embodiments, the combination may be used to treat, prevent, reduce the incidence, reduce symptoms, and/or reduce recurrences of non-alcoholic fatty liver disease (NAFLD) and/or any other liver disorder in a subject.

Sulforaphane is also known as 1-isothiocyanate-4-methylsulfinylbutane. Sulforaphane derivatives include, but are not limited to, sulfoxythiocarbamate analogs of sulforaphane, 6-methylsulfinylhexylisothiocyanate (6-HITC), and compounds containing a sulforaphane structure with different side chains and/or different spacer lengths between the isothiocyanate and sulfoxide groups. Examples of sulforaphane derivatives include those described in the following references, each of which is incorporated herein by reference: 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, 201 1, 85(4):241-272.

In some embodiments of the invention, the composition contains sulforaphane or its derivative, preferably sulforaphane, in an amount of from 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, compositions suitable for use in humans contain about 1 mg to about 20 mg.

In some embodiments, the methods of the present invention comprise administering to the subject sulforaphane or a derivative thereof, preferably sulforaphane, in an amount of 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 of the invention in which the subject is a human, the method comprises administering from about 1 mg to about 20 mg. In some embodiments, the methods of the present invention comprise administering to the 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 certain preferred embodiments where the subject is a human, the method comprises administering about 2 μg/kg to about 2 mg/kg, and more preferably about 0.01 mg/kg to about 0.3 mg/kg. The above amounts may refer to a single dose or a total daily dosage. By total daily dosage is meant the total amount of a compound or ingredient administered to a subject in twenty-four hours.

In some embodiments, the compositions contain more than one sulforaphane or derivative thereof. For example, the methods or composition may contain both sulforaphane and one or more of its derivatives, or two or more of its derivatives. In some embodiments of the invention, in which the method or composition contains more than one sulforaphane or its derivative, the above amounts may refer to the amount of sulforaphane or its derivative or the total amount of more than one sulforaphane or its derivative.

The term "sulforaphane precursor" refers to any compound, substance, or material that can be used to make sulforaphane. In preferred embodiments, the sulforaphane precursor contains a compound that can be converted or preferably enzymatically metabolized to sulforaphane. In some preferred embodiments, the sulforaphane precursor contains glucoraphanin.

Glucoraphanin is a glucosinolate, also known as 4-methylsulfinylbutyl glucosinolate and 1-S-[(1E)-5-(methylsulfinyl)-N-(sulfonatooxy)pentanimidoyl]-1-thio-β-D-glucopyranose.

In some embodiments of the invention, 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, compositions suitable for use in humans contain about 3.5 mg to about 50 mg of a sulforaphane precursor, preferably glucoraphanin.

In some embodiments, the method comprises administering a sulforaphane precursor, preferably glucoraphanin, to the subject in an amount of 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. 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 a 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 where the subject is a human, the method comprises administering about 50 µg/kg to about 800 µg/kg.

The above amounts may refer to a single dose or a total daily dosage.

In some embodiments, the method comprises administering more than one sulforaphane precursor. In some embodiments, the composition contains more than one sulforaphane precursor. In some embodiments of the invention, in which the method or composition contains more than one sulforaphane precursor, the above amounts may refer to the amount of each sulforaphane precursor or the total amount of sulforaphane precursors.

The sulforaphane precursor can be converted or metabolized to sulforaphane. In some embodiments, the sulforaphane precursor is enzymatically converted to sulforaphane. In some embodiments, the enzyme capable of converting a sulforaphane precursor to sulforaphane comprises a glucosidase enzyme, preferably a thioglucosidase enzyme, and more preferably a myrosinase. Myrosinase is also known as thioglucoside glucohydrolase.

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 use in humans contain about 5 ng to about 75 ng of the enzyme.

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 of the invention, in which the subject is a human, the method comprises the introduction of about 5 ng to about 75 ng of the enzyme. In some embodiments, the method comprises administering the enzyme to the subject in an amount 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 up to about 2 ng/kg. In some preferred embodiments of the invention, in which 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 a single dose or a total daily dosage.

In some embodiments, the method comprises administering more than one enzyme capable of converting a sulforaphane precursor to sulforaphane.

In some embodiments, the composition contains more than one enzyme capable of converting a sulforaphane precursor to sulforaphane. In some embodiments of the invention, in which the methods or compositions contain more than one enzyme, the above amounts may refer to the amount of each enzyme or the total number of enzymes.

The present invention also provides the use of broccoli extract and/or powder, including, but not limited to, broccoli seed and sprout extracts and powders. The present invention provides methods for administering broccoli extract and/or powder and compositions containing broccoli extract and/or powder. In some embodiments of the invention, the extract or powder of broccoli is standardized to a content of from about 1 wt.% to about 75 wt. %, more preferably about 2.5 wt.% up to about 50 wt. %, even more preferably about 5 wt.% to about 25 wt.% and most preferably about 10 wt.% to about 20 wt.% precursor sulforaphane, preferably glucoraphanin. Examples of broccoli extracts and powders include, but are not limited to, those described in US 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 herein in its entirety by reference. Broccoli powders can be obtained, for example, by air drying, freeze drying, tumble drying, spray drying, thermal drying and/or low vacuum drying of broccoli, preferably broccoli sprouts. In some embodiments, the compositions and methods comprise 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 amounts sufficient to provide the subject with the sulforaphane precursor or sulforaphane in the amounts described above. In some embodiments, the composition may further comprise an enzyme potentiator, preferably ascorbic acid. In some embodiments, the method may further comprise administering an enzyme potentiator, preferably ascorbic acid.

Sulforaphane or a derivative thereof, a sulforaphane precursor, and/or an enzyme capable of converting a sulforaphane precursor to sulforaphane can be obtained from any source, including, but not limited to, one or more of the Brassicaceae (also known as Cruciferae) plants. Examples of plants in the Brassicaceae family include, but are not limited to, broccoli, Brussels sprouts, sparassis crispa, cabbage, horseradish, parsnip, radish, Japanese horseradish, field mustard, and white mustard. In some preferred embodiments, the sulforaphane precursor, preferably glucoraphanin and the enzyme, preferably myrosinase, are derived from broccoli, broccoli sprouts, or broccoli seeds. The sulforaphane precursor and enzyme may be obtained from the same source or from different sources. In some embodiments, the sulforaphane precursor and enzyme can be obtained from an extract or powder of these plants, preferably an extract or powder of broccoli seeds or sprouts.

The present invention provides the use of an enzyme potentiator. Enzyme potentiators can be used to increase the activity of an enzyme that promotes the conversion of the sulforaphane precursor to sulforaphane. In some embodiments, the enzyme potentiator contains an enzyme cofactor, preferably ascorbic acid. Ascorbic acid, also known as ascorbate or vitamin C, can potentiate myrosinase activity. In some embodiments of the invention, without an enzyme potentiator such as ascorbic acid, the conversion reaction to sulforaphane may proceed too slowly for the localization required for maximum absorption. The enzyme potentiator can be obtained from a natural source or it can be obtained synthetically.

In some embodiments, the compositions may contain about 1 mg to about 500 mg, preferably about 1 mg to about 250 mg, and most preferably about 1 mg to about 125 mg of an enzyme potentiator. In some preferred embodiments of the invention, compositions suitable for use in humans contain about 1 mg to about 50 mg of an enzyme potentiator.

In some embodiments, the method of the present invention comprises administering an enzyme potentiator, preferably ascorbic acid, in an amount of about 1 mg to about 500 mg, preferably about 1 mg to about 250 mg, and most preferably about 1 mg to about 125 mg. In certain preferred embodiments where the subject is a human, the method comprises administering about 1 mg to about 50 mg. In some embodiments, the method of the present invention comprises administering an enzyme potentiator, preferably ascorbic acid, in an amount of about 0.01 mg/kg to about 3 mg/kg, and preferably about 0.02 mg/kg to about 2 mg/kg . In certain preferred embodiments where the subject is a human, the method comprises administering about 0.02 mg/kg to 0.7 mg/kg of an enzyme potentiator. The above amounts may refer to a single dose or a total daily dosage.

In some embodiments, the method comprises administering more than one enzyme potentiator. In some embodiments of the invention, the composition contains more than one enzyme potentiator. In some embodiments of the invention, in which the method or composition contains more than one enzyme potentiator, the above amounts may refer to the amount of any enzyme potentiator or the total number of enzyme potentiators.

The present invention also includes the use of milk thistle extract or powder. The milk thistle belongs to the species Silybum marianum. Milk thistle contains a number of components or fractions with biological activity. The active fraction of milk thistle is silymarin, which contains a number of components.

Examples of silymarin components include, but are not limited to: silybinin (also known as silybin), isosilybinin, silicristin, silydianin and quercetin, dehydrosilybin, deoxysilicystin, deoxysilydianin, silandrin, silybinome, silygermine, and neosilygermine. Silibinin isomers include Silibinin A and Silibinin B. In preferred embodiments, milk thistle extract or powder contains silymarin.

In some embodiments, the compositions and methods of the present invention may comprise the use of one or more silymarin derivatives, instead of or in addition to milk thistle extract or powder. Silymarin derivatives include any modified forms of the above compounds, including, but not limited to, 7-O- and 23-O-acyl derivatives and analogs. Examples of silymarin derivatives include, but are not limited to, 2,3-dehydrosilybin (DHS); 7-O-methylsilybin; 7-O-galloylsilybin; 7,23-silybin disulfate (DSS); 7-O-palmitoylsilybin; and 23-O-palmitoylsilybin. Examples of derivatives include those described in the following references, each of which is incorporated herein by reference: Agarwal et al. FLOS ONE, 2013, 8(3):e60074; GB 2167414 and CA 1337124. In some embodiments, the compositions and methods of the present invention may comprise the use of silymarin or silibinin in purified form, or synthetically prepared silymarin or silibinin, instead of or in addition to milk thistle extract or powder.

In some embodiments of the invention, milk thistle extract or powder may be used. In preferred embodiments, the milk thistle extract contains silymarin. In preferred embodiments, the milk thistle extract contains silibinin. In some embodiments, the milk thistle extract or powder is standardized to contain about 25% to about 95%, preferably about 50% to about 90%, and more preferably about 55% to about 85% silymarin. In some embodiments, the milk thistle extract or powder is standardized to contain about 5% to about 75%, preferably about 10% to about 60%, more preferably about 15% to about 50%, and most preferably about 20% to about 35% silibinin. . Examples of milk thistle extract include, but are not limited to, those described in US Patent No. 6,555,141; US Patent No. 6,863,906; US Patent No. 7563779; application WO 200908006; application EP 2020238; WO 2009043671; patent EP 1584240 and application WO 2011076154, each of which is incorporated herein in its entirety by reference. Milk thistle powders can be obtained, for example, by air drying, freeze drying, tumble drying, spray drying, thermal drying and/or low vacuum drying of milk thistle.

In some embodiments, the compositions and methods comprise the use of about 1.25 mg to about 15 grams, preferably about 5 mg to about 10 grams, and most preferably about 10 mg to about 7.5 grams of milk thistle extract. In some preferred embodiments of the invention, in which the composition is suitable for use in humans, the composition contains about 25 mg to about 5 grams of milk thistle. In some embodiments, the composition contains about 0.75 mg to about 15 grams, preferably about 3 mg to about 7 grams, more preferably about 7 mg to about 5 grams, and most preferably about 15 mg to about 3.5 grams of silymarin. In some preferred embodiments of the invention, in which the composition is suitable for use in humans, the composition contains about 50 mg to about 200 mg of silymarin. In some embodiments, the composition contains about 0.3 mg to about 5 grams, preferably about 1.5 mg to about 3 grams, more preferably about 3 mg to about 2 grams, and most preferably about 7 mg to about 1.5 grams of silibinin. . In some preferred embodiments of the invention, in which the composition is suitable for use in humans, the composition contains about 30 mg to about 90 mg of silibinin.

In some embodiments, the method comprises administering silymarin in an amount of about 1 µg/kg to about 75 mg/kg, preferably about 2.5 µg/kg to about 50 mg/kg, more preferably about 5 µg/kg to about 25 mg /kg, more preferably about 10 µg/kg to about 15 mg/kg, and most preferably about 15 µg/kg to about 10 mg/kg. In certain preferred embodiments where the subject is a human, the method comprises administering about 1 mg/kg to about 3 mg/kg of silymarin. In some embodiments, the method comprises administering silibinin in an amount of about 0.5 µg/kg to about 50 mg/kg, preferably about 1 µg/kg to about 35 mg/kg, more preferably about 2.5 µg/kg to about 25 mg/kg, more preferably about 5 µg/kg to about 10 mg/kg, and most preferably about 7.5 µg/kg to about 5 mg/kg. In some preferred embodiments of the invention, in which the subject is a human, the method further comprises administering about 300 μg/kg to about 2 mg/kg of silibinin. In embodiments that use a derivative of the silymarin component, the methods and compositions may contain the derivative in an amount of about 0.75 mg to about 15 grams, preferably about 3 mg to about 7 grams, more preferably about 7 mg to about 5 grams, and most preferably about 15 mg to about 3.5 grams. In some preferred embodiments of the invention, in which the composition is suitable for use in humans, the compositions contain about 50 mg to about 200 mg of a derivative of the silymarin component. The above amounts may refer to a single dose or a total daily dosage.

In some embodiments of the invention, S-adenosylmethionine can be used instead of or in addition to milk thistle extract, silymarin and/or silibinin.

The methods of the present invention may also contain the introduction of one or more additional components.

The compositions of the present invention may also contain one or more additional components. Additional components may include active pharmaceutical ingredients, nutritional supplements and nutritional extracts. Examples of additional components include, but are not limited to, quercetin or a derivative thereof, amino sugar such as glucosamine, glycosaminoglycan such as chondroitin, avocado and soybean unsaponifiables, vitamins such as vitamin K2, coffee tree fruit, magnesium, ursolic acid proanthocyanidins, alpha and beta glucans, curcumin, phytosterols, phytostanols and S-adenosylmethionine (SAMe). These additional components can be found in cranberry (Vaccinium macrocarpon) extract (proanthocyanidins, quercetin and ursolic acid), turmeric (Curcuma longa), medicinal mushroom extracts such as shiitake (Lentinus edodes), curly mushroom (Grifola frondosa) and lacquered tinder (Ganoderma lucidum).

In some embodiments, the ratio of silymarin to sulforaphane or derivative thereof is about 1:1 to about 75:1, more preferably about 2:1 to about 50:1, more preferably about 2.5:1 to about 25:1, still more preferably about 5:1 to about 15:1 and most preferably about 6:1 to about 9:1. In some embodiments, the ratio of silibinin to sulforaphane or derivative thereof is about 1:2 to about 35:1, more preferably about 1:1 to about 25:1, more preferably about 1:1 to about 15:1, even more preferably about 2:1 to about 10:1, and most preferably about 2:1 to about 5:1. In some embodiments, the ratio of silymarin to sulforaphane precursor is about 1:5 to about 50:1, preferably about 1:2 to about 25:1, more preferably about 1:1 to about 10:1, more preferably about 1.5 :1 to about 5:1 and most preferably about 1:1 to about 4:1. In some embodiments, the ratio of silibinin to sulforaphane precursor is about 1:5 to about 50:1, preferably about 1:2 to about 25:1, preferably 1:1 to about 20:1, and most preferably about 1:1 to about 13:1.

In some embodiments of the invention, the composition contains a single 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 of the invention, the composition comprises an oral dosage form or a rectal dosage form. Examples of dosage forms for oral administration include, but are not limited to, a tablet, a capsule, a powder that can be dispersed in a beverage, a liquid such as a solution, suspension, or emulsion, a soft chewable gel capsule, a chewable lozenge, or other convenient dosage form, known in the art. In preferred embodiments, the composition comprises a tablet, capsule or soft chewable treat. Dosage forms for oral administration may be formulated for immediate release, delayed release, or sustained release.

In some embodiments, at least the sulforaphane precursor, enzyme, and enzyme potentiator are provided in a dosage form allowing release in an area of the gastrointestinal tract having a pH of at least 4 and preferably at least 5, such as the small intestine, preferably in the duodenum. In some embodiments of the invention, at least the sulforaphane or its derivative and/or extract or powder of broccoli is provided in a dosage form allowing release in the area of the gastrointestinal tract, having a pH of at least 4 and preferably at least 5, such like the small intestine, preferably in the duodenum. In some embodiments of the invention, milk thistle extract or powder and/or any optional additional components are also released in the gastrointestinal tract at a pH of at least 4 and preferably at least 5, such as the small intestine, preferably in the duodenum. gut. The small intestine includes the duodenum, jejunum, and ileum.

In some embodiments, each of these components (i.e., sulforaphane precursor, enzyme, enzyme potentiator, sulforaphane or derivative thereof, broccoli extract or powder, milk thistle extract or powder, and/or additional components) are released simultaneously or concomitantly with one another (then eat for a short period of time one after the other). This provides advantages over glucoraphanin-containing compositions formulated to release glucoraphanin in the gastrointestinal tract at a pH below 4, such as the stomach. In low pH environments, such as the one just mentioned, an acidic environment can promote the conversion of the sulforaphane precursor to other physiologically inactive end products such as sulforaphane nitrile and epithionitrile.

In some embodiments, the compositions may include compositions for oral administration containing gastroprotective formulations, including enteric-coated dosage forms or any dosage forms that are resistant to disintegration in a region of the gastrointestinal tract having pH values below 4, such as the stomach. For example, the composition for oral administration may contain a tablet or capsule containing an enteric coating. The enteric coating may contain materials including, but not limited to, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, polyvinyl acetate phthalate, methacrylic acid copolymer, methacrylic acid-acrylic ester copolymer, hydroxypropyl methylcellulose acetate succinate, hydroxymethylpropyl methylcellulose trimellitate, shellac, cellulose acetate trimellitate, carboxymethyl ethyl cellulose, and mixtures thereof.

The enteric coating may contain any suitable enteric polymers known in the art. In some embodiments of the invention, one or more components of the composition may be incorporated into the matrix of enteric polymers. In some embodiments, compositions for oral administration comprise a capsule that dissolves slowly in gastric acid and enters the small intestine, such as DRCAPS™ acid-resistant capsules sold under the name ^CAPSUGEL® or any other acid-resistant capsules. In the most preferred form, the composition for oral administration is surrounded by a coating that is insoluble until the environmental pH is greater than at least 4 and more preferably at least 5. In other words, a controlled release coating can be used as counteracting the pH, at a rate adjusted so that the components are not released until the pH of the gastrointestinal tract increases to at least 4 and more preferably at least 5. Thus, for the purpose of protection a sulforaphane precursor, an enzyme capable of converting a sulforaphane precursor to sulforaphane; and an enzyme or sulforaphane potentiator in the gastric environment, a sustained release dosage form may be used. The coating layer(s) can be applied to the oral composition using standard application techniques. Enteric coating materials can be dissolved or dispersed in organic or aqueous solvents. The pH at which the enteric coating dissolves can be controlled by the choice of polymer or combinations of polymers and/or the ratio of side groups. For example, the solubility parameters of the polymer film can be changed by the ratio of free carboxyl groups to ester groups. The enteric coating layers also contain pharmaceutically acceptable plasticizers such as triethyl citrate, dibutyl phthalate, triacetin, polyethylene glycols, polysorbates, or other plasticizers. Adjuvants such as dispersants, colorants, release agents and antifoam agents may also be included.

The compositions may contain one or more of the inactive pharmaceutical ingredients (also commonly known as "excipients"). Inactive ingredients, for example, serve to solubilize, suspend, thicken, dilute, emulsify, stabilize, preserve, protect, color, flavor, i.e. transform the active ingredients into a usable and effective preparation that is safe, convenient to use, in other words, acceptable to use the remedy. The excipients are preferably pharmaceutically acceptable excipients. Exemplary classes of pharmaceutically acceptable excipients include lubricants, buffering agents, stabilizers, foaming agents, pigments, colorants, flavoring agents, additives, fillers, flavoring agents, release modifiers, adjuvants, plasticizers, flow accelerators, lubricating agents, polyols, granulating agents, diluents, binders, buffers, absorbents, lubricants, adhesives, antiblocking agents, acidifiers, softeners, resins, soothing agents, solvents, surfactants, emulsifiers, elastomers and mixtures thereof.

In some embodiments, a 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 potentiator, preferably an enzyme cofactor, more preferably ascorbic acid and (iv) milk thistle extract or powder shows a synergistic effect. In some embodiments, the combination of sulforaphane (or a derivative thereof) and milk thistle extract or powder exhibits a synergistic effect. Synergy means an effect in which the combination of two or more components provides a result that is greater than the sum of the effects due to the agents taken separately. In preferred embodiments of the invention, the synergistic effect outweighs the additive effect. In some embodiments, the combination of a sulforaphane precursor, an enzyme capable of converting the sulforaphane precursor to sulforaphane, an enzyme potentiator, and milk thistle extract or powder has a statistically significant effect that is superior to: (i) each component taken alone, (ii) the combination of the precursor sulforaphane and enzyme taken separately; and/or (iii) a combination of a sulforaphane precursor, an enzyme, and an enzyme potentiator taken separately.

In preferred embodiments, the combination of sulforaphane precursor, enzyme, enzyme potentiator and milk thistle extract or powder exhibits synergy, having a statistically significant and/or superior additive effect compared to sulforaphane precursor alone and milk thistle extract or powder alone. In some embodiments of the invention, the combination of glucoraphanin, myrosinase, ascorbic acid and silymarin has a synergistic effect compared to the combination of glucoraphanin, myrosinase, ascorbic acid taken alone and compared to silymarin taken alone. In some embodiments of the invention, the combination of glucoraphanin, myrosinase, ascorbic acid and silibinin has a synergistic effect compared to the combination of glucoraphanin, myrosinase, ascorbic acid taken alone and compared to silibinin taken alone.

In some embodiments, the combination of sulforaphane (or a derivative thereof) and milk thistle extract or powder has a statistically significant and/or greater additive effect than: (i) sulforaphane (or a derivative thereof) alone and/or (ii) an extract or milk thistle powder taken separately. In some embodiments, the combination of sulforaphane and silymarin has a synergistic effect compared to sulforaphane alone and silymarin alone. In some embodiments, the combination of sulforaphane and silibinin has a synergistic effect compared to sulforaphane alone and silibinin alone.

In some embodiments, the combination of broccoli extract or powder and milk thistle extract or powder has a statistically significant and/or superior additive effect compared to: (i) broccoli extract or powder alone, and/or (ii) milk thistle alone. In some embodiments, the combination of broccoli extract or powder and silymarin has a synergistic effect compared to broccoli extract or powder alone and silymarin alone. In some embodiments, the combination of broccoli extract or powder and silibinin has a synergistic effect compared to broccoli extract or powder alone and silibinin alone.

The present invention provides methods of use, including methods of administration to a subject in need thereof. In some embodiments, the method comprises administering a combination of a sulforaphane precursor, an enzyme capable of converting the sulforaphane precursor to sulforaphane, an enzyme potentiator, and milk thistle extract or powder. In some embodiments, the method comprises administering a combination of sulforaphane or a derivative thereof and milk thistle extract or powder. In some embodiments, the method comprises administering a combination of broccoli extract or powder and milk thistle extract or powder.

In some embodiments of the invention, the method relates to the treatment, prevention, reduction in frequency, reduction of symptoms, and / or reduction of recurrence of a disease or condition associated with the liver, genitourinary system (including the prostate, breast and ovaries), brain, lungs, kidneys , colon, esophagus, pancreas, or hematopoietic system in a subject, comprising administering to the subject. The methods may be useful in reducing or slowing damage to tissues and organs such as the liver, genitourinary system (including the prostate, breast and ovaries), brain, lungs, kidneys, colon, esophagus, and pancreas. In some embodiments of the invention, the method relates to increasing the content of glutathione in a subject in need of it. The method may also be useful in the treatment, prevention, symptom reduction, and/or reduction of recurrence of diseases or conditions associated with low glutathione levels.

Examples of such diseases and conditions include, but are not limited to, non-alcoholic fatty liver disease (NAFLD), cancer (such as cancer of the liver, lung, prostate, colon, breast, brain, ovary, esophagus, pancreas, nasopharynx, osteosarcoma ), leukemia, cystic fibrosis, HIV, glutathione synthetase deficiency, Alzheimer's disease, Parkinson's disease, Huntington's disease, Charcot's disease, Friedreich's hereditary ataxia, multiple sclerosis, fibromyalgia, chronic fatigue, autism and diabetes, hepatotoxicity and environmental toxicity.

In some embodiments, the method relates to the treatment, prevention, reduction in incidence, reduction in symptoms, and/or reduction in recurrence of non-alcoholic fatty liver disease (NAFLD) and/or any other liver disorder in a subject. In some embodiments, the methods relate to improving or reducing the harmful effects of NAFLD.

In some embodiments of the invention, the methods relate to providing a positive effect on biomarkers and treatment, prevention, reduction in frequency, reduction of 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-3-transphrase theta 2 (GSTT2), microsomal glutathione-S-transferase 1 (MGST1), aldehyde oxidase (AOX1), aldokete reductase 1 B8 (Akrl b8), flavin-containing monooxygenase 2 ( FMO2), Fc receptor region III receptor (Fcgr3), tryptase beta 1 (TPSB1), mast cell protease-6 (Mcpt6), neurexin-1-alpha (NRXN-1), microphthalmia-associated transcription factor (MITF), iodothyronine deiodinase II (DIO2), angiopoietin-14 (Angpt14), cluster of differentiation (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 intervillositis placenta, hypertension, abdominal aortic aneurysm, inflammatory bowel disease, chronic rhinosinusitis, coronary artery disease, and kidney disease.

In some embodiments, the method comprises administering to a subject in need thereof a combination of sulforaphane and milk thistle extract or powder. In some embodiments, the method comprises administering to a subject in need thereof a combination of broccoli extract or powder and milk thistle extract or powder. In some preferred embodiments, the method comprises administering to the subject a combination of glucoraphanin, myrosinase, ascorbic acid, and milk thistle extract or powder. In preferred embodiments of the invention, the combinations in the methods of the present invention show a synergistic effect.

In preferred embodiments, one or more component combinations (e.g., a sulforaphane precursor, an enzyme capable of converting a sulforaphane precursor to sulforaphane, an enzyme potentiator, milk thistle extract or powder or sulforaphane or a derivative thereof, and milk thistle extract or powder or broccoli extract or powder, and milk thistle extract or powder) are administered together in the same composition or dosage form, or separately, preferably during the overlapping time of their therapeutic properties. In some embodiments of the invention, the components of the combinations can be used in two or more compositions or dosage forms administered orally. For example, in some embodiments, the sulforaphane precursor, an enzyme capable of converting the sulforaphane precursor to sulforaphane, and the enzyme potentiator are administered in an orally administered dosage form, while the milk thistle extract or powder is administered in one or more separate or additional s) dosage form(s). In preferred embodiments, the components of the combination are administered in a single dosage form.

In some embodiments, the combination may be administered at a frequency of 1 to 10 times per day, preferably 1 to 5 times per day, more preferably 1 to 3 times per day, and most preferably 1 time per day.

The dosages disclosed in this application generally refer to dosages applicable to humans. Dosage calculations can be determined by those skilled in the art, taking into account body weight, surface area, metabolic rate, and species differences.

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, seals, and rabbits. In preferred embodiments of the invention, the subjects are mammals that are not eaten, such as humans, cats and dogs.

EXAMPLES

Example 1

The following is an exemplary recipe:

Broccoli extract containing glucoraphanin (about 12 wt.%), 50 mg to 5 g

Freeze-dried powder of broccoli sprouts containing myrosinase, 25 mg to 500 mg

Ascorbic acid, 5 mg to 500 mg

Milk thistle extract (about 20 wt.% to 35 wt.% silibinin), 25 mg to 5 g.

Example 2

A Hydrophobic Interaction Chromatography (HILIC) method has been developed that meets the following conditions: Column: Waters BEN Amide, particle size 1.7 µm; 2.1mm×100mm

Mobile phase: 20% 10 mM ammonium acetate, pH 5.0; 80% acetonitrile;

Separation method: isocratic

Column temperature: 70°C

Flow rate: 0.7 ml/min

The above conditions allow separation of five typical Brassicaceae glucosinolates, including the sulforaphane precursor, glucoraphanin.

Example 3

Glucoraphanin consumption as a function of ascorbic acid concentration.

About 250 mg of a broccoli seed extract containing about 12% by weight of glucoraphanin was hydrolyzed at a fixed concentration of myrosinase from broccoli sprouts in the presence of ascorbic acid having a concentration ranging from 0 to 600 µmol/l. The reaction mixtures were thermostated at 38°C; aliquots were taken every 15 minutes for 60 minutes and chromatographically determined the concentration of glucoraphanin. The rate of consumption of glucoraphanin was interpreted as the rate of its conversion to sulforaphane. The graphic representation of the decrease in the content of glucoraphanin depending on the increase in the concentration of ascorbic acid is presented as a series of linear curves; the tangents of the slope of the linear regression lines reflect the rate of consumption of glucoraphanin in µmol/minute. It can be seen that in the presence of ascorbic acid with a concentration of 600 µmol/liter, the reaction rate increases by 13 times, compared to what it would have been without the modulating effects of ascorbic acid.

Example 4

Equimolar conversion of glucoraphanin to sulforaphane.

To further elucidate the role of ascorbic acid in modulating myrosinase activity, a two-part experiment was performed. All solutions were prepared in 20 mM Tris (tris-hydroxymethylaminomethane) buffered saline at pH 7.5, which was previously shown to be optimal for myrosinase activity; an accurate weighing of 100 mg of lyophilized broccoli powder was placed in each test tube as a source of myrosinase. The experiment was carried out at 38° C. for 2 hours, aliquots of the sample were taken at 30-minute intervals and the content of both glucoraphanin and sulforaphane was determined using HPLC (high performance liquid chromatography). In order to instantly inhibit further myrosinase activity, a strongly acidic "stop" solution was used in selected aliquots. The control sample did not contain ascorbic acid, and the enzymatic conversion proceeded without the participation of the cofactor.

PART 1 In the presence of ascorbic acid at a constant concentration of 1 mmol/liter, broccoli seed extract (about 12 wt.% glucoraphanin) was added, increasing its amount from 250 mg to 500 mg.

PART 2. With a fixed amount of broccoli seed extract equal to 250 mg, the concentration of ascorbic acid was varied from 0.4 mmol/liter to 3.8 mmol/liter.

The table below shows the concentrations of glucoraphanin and sulforaphane expressed in micromoles. It can be seen that during the first 30 minutes, in almost all reaction mixtures, glucoraphanin was completely converted to sulforaphane. However, a careful study of the enzymatic conversion in the control sample, without the stimulatory effects of ascorbic acid, reveals an equimolar conversion of glucoraphanin to sulforaphane, i.e., the amount of glucoraphanin consumed is equivalent to the amount of sulforaphane produced.

In part 2 of the experiment, the modulating effect of increasing the concentration of ascorbic acid on the activity of myrosinase was studied. An initial, apparently linear increase in the conversion of glucofaranin to sulforaphane, stimulated by myrosinase, was observed until an ascorbic acid concentration of 2 mmol/l was reached, and was subsequently replaced by a significant leveling.

In conclusion, a study of the yield of sulforaphane after 30 minutes of the Part 1 experiment revealed that in the presence of 1 mmol/liter of ascorbic acid, a fixed amount of myrosinase contained in 100 mg of freeze-dried broccoli sprout powder, according to a predictable linear law, is able to generate at least 200 µmol sulforaphane. FIG. 1, 2, 3 and 4 illustrate the results of this study.

Example 5

The conversion of glucofaranin to sulforaphane in the presence of a fluid that mimics intestinal juice.

A powder of liquid imitating intestinal juice (SIF), a commercially available concentrate, similar in composition, pH and ionic strength of the solution to the contents of the human intestine, was used. The USP dissolution apparatus 2 (paddle) was used in the experiment, where 500 ml of fluid simulating intestinal juice was placed in six dissolution vessels together with 150 mg of lyophilized broccoli sprout powder as a source of myrosinase. In vessels 1-4, the concentration of ascorbic acid varied from 0.25 to 1.00 mmol/l; in vessel 5, in addition to 1 mmol/l of ascorbic acid, 3.125 g of pancreatin (8xUSP) were suspended; in vessel 6, in addition to 1 mmol/l of ascorbic acid and 3.125 g of pancreatin (8xUSP), was added twice the amount of lyophilized powder of broccoli sprouts (300 mg). After bringing the temperature of the vessels to 38°C, 250 mg of broccoli seed extract rich in glucoraphanin (12 wt.%) was added to each vessel, and the resulting suspensions were stirred at 75 rpm for 2 hours. Aliquots were taken every 15 minutes and analyzed for sulforaphane. FIG. 4 shows a direct relationship between an increase in the yield of sulforaphane and an increase in the concentration of ascorbic acid, especially in the early stages of the experiment.

Example 6

The following study was performed to determine the effect of the combination of sulforaphane and silibinin on the content of glutathione. Glutathione plays an important role in the body as it can serve as an antioxidant, detoxifier, and immune booster. Reduced levels of glutathione can make the patient susceptible to oxidative stress, disease, and cancer. Therefore, increasing the content of glutathione produces a positive effect.

In the study, a HepG2 human liver cancer cell line was treated with DMSO (solvent control), sulforaphane (SFN), silibinin (Silib) or a combination of sulforaphane and silibinin for 24 hours. Cell lysates were collected and glutathione content was measured using o-phthalaldehyde (ORT) as a fluorescent reagent. FIG. 5 and FIG. 6 show the results of the study.

In part 1 of the study, the effect of 0.5 µm SFN was compared with the effect of various concentrations of silibinin and with the combination of 0.5 µm SFN and various concentrations of silibinin. Specifically, cells were treated with one of the following: (i) DMSO (solvent control), (ii) 0.5 µm SFN, (iii) 100 µm Silib, (iv) 200 µm Silib, (v) 300 µm Silib, (vi) 0.5 µm SFN and 100 µm Silib, (vii) 0.5 µm SFN and 200 µm Silib, and (viii) 0.5 µm SFN and 300 µm Silib. The results show that the combination of sulforaphane and silibinin at each of the tested doses has a synergistic effect compared to each of the components taken separately. For example, when cells were treated with the individual components, the glutathione content remained the same when treated with sulforaphane alone and decreased slightly when treated with silibinin, compared to the DMSO control (solvent control). However, when the cells were treated with the combination of sulforaphane and silibinin, at each of the doses tested, the glutathione content increased synergistically compared to the control. An increase in the content of glutathione produces a positive effect. The results are shown in FIG. five.

In part 2 of the study, the effect of 2 µm SFN was compared with the effect of various concentrations of silibinin and the combination of 2 µm SFN and various concentrations of silibinin. Specifically, cells were treated with one of the following: (i) DMSO (solvent control), (ii) 2 µm SFN, (iii) 100 µm Silib, (iv) 300 µm Silib, (v) 2 µm SFN and 100 µm Silib, and ( vi) 0.5 µm SFN and 300 µm Silib. The results show that the combination of sulforaphane and silibinin at each of the tested doses has a synergistic effect compared to each component taken separately. For example, when cells were treated with individual components, the glutathione content remained the same when treated with sulforaphane taken alone, or slightly decreased when treated with silibinin compared to control. However, when the cells were treated with the combination of sulforaphane and silibinin, at each of the doses tested, the glutathione content increased synergistically compared to the control. Increasing the glutathione content produces a positive effect through cell detoxification. The results are shown in FIG. 6.

Example 7

The subject has been diagnosed with non-alcoholic fatty liver disease (NAFLD) and has symptoms including malaise, fatigue, and abdominal discomfort. She took a tablet containing glucoraphanin, myrosinase, ascorbic acid, and milk thistle extract.

The tablet is an enteric dosage form that releases the contents in the small intestine. After one month of taking the tablet daily, the subject experienced modulation of surrogate biomarkers, including glutathione, which correlates with improvement in symptoms.

Claims (8)

1. Oral composition for increasing glutathione in the treatment of cancer selected from liver cancer, rectal cancer and colon cancer, containing a combination of sulforaphane and milk thistle extract standardized to contain from 20 to 35 wt.% silibinin. 2. An oral composition according to claim 1, wherein the composition is an enteric dosage form. 3. An oral composition according to claim 1, further comprising one or more additional components selected from the group consisting of: quercetin, amino sugar, glycosaminoglycan, avocado and soybean unsaponifiables, vitamin, coffee tree fruit, magnesium, silymarin , proanthocyanidins, ursolic acid, curcumin, phytostyrenes and phytostanols. 4. An oral composition according to claim 1, wherein the composition further comprises a broccoli extract standardized to contain 12 wt% glucoraphanin. 5. An oral composition for increasing glutathione in the treatment of cancer selected from liver cancer, rectal cancer and colon cancer, comprising a combination of broccoli extract or powder standardized to 10 to 20 wt % glucoraphanin and milk thistle extract , standardized to contain from 20 to 35 wt.% silibinin. 6. An oral composition according to claim 5, wherein the composition is an enteric dosage form. 7. An oral composition according to claim 5, further comprising one or more additional components selected from the group consisting of: quercetin, amino sugar, glycosaminoglycan, avocado and soybean unsaponifiables, vitamin, coffee tree fruit, magnesium, silymarin , proanthocyanidins, ursolic acid, curcumin, phytostyrenes and phytostanols. 8. An oral composition according to claim 5 wherein the broccoli extract or powder is standardized to contain 12% by weight glucoraphanin.

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