THERAPEUTIC USES OF AN ANTI-CANCER COMPOSITION DERIVED FROM RICE BRAN

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application is a CIP of U.S. Application Nos. 11/375,41 1, filed March 13, 2006 and 1 1/677,018 filed February 20, 2007. The disclosures of which are hereby incorporated by reference in their entireties for all purposes.

BACKGROUND OF THE INVENTION [0002] Dietary fiber is a heterogeneous group of substances which have only one common characteristic: the non-digestibility in the small bowel. With one exception, all fibers are non-caloric carbohydrate polymers (poly- or disaccharides). Some fibers are water-soluble, others are insoluble. This property is associated with physiological effects. Soluble (viscous) fibers can bind water and thus form hydrocolloids or gels, insoluble fibers cannot form gels.

[0003] Dietary fibers also play an essential role in the physiology of the gastrointestinal tract. They modify the absorption of nutrients (particularly carbohydrates and lipids) in the small bowel. They accelerate the gut transit time and determine stool composition and quantity. They are the main nutritional source for the colonic microflora. During the bacterial fermentation short-chain fatty acids are formed which are essential for nutrition and integrity of the colonocytes and for colonic function. Moreover gases, detoxifying enzymes, antioxidants and carcinogen-inactivating compounds arise. The most important fibers are cellulose, hemicellulose, pectin, guar, psyllium, beta-glucan, lignin and digestion-resistant starch; they are present in varying amounts in plant foods and in fiber preparations. The usual daily intake of dietary fiber in Europe and the United States amounts to only 15-20 grams, while health authorities and nutrition societies recommend a reference value of at least 30 grams per day. Dietary fibers are applied as food-integrated substances, as supplements to be taken with food, and as purified substances.

[0004] The health benefits of dietary fiber are associated with a decreased incidence of certain diseases such as cardiovascular disease, diabetes, cancer of several organs, kidney stones as well as an improvement in colon health. Although certain dietary fiber

formulations are available, a need exists for formulations with a practical shelf-life and which maximizes the benefits of fiber to consumers of such formulations.

SUMMARY OF THE INVENTION

[0005] The present invention provides a stabilized rice bran fiber and methods of treating or preventing various maladies through the consumption or administration of the fiber.

[0006] In one embodiment, the invention provides a rice bran fiber composition derived from pre-digested rice bran, wherein the composition comprises soluble rice bran fiber, insoluble rice bran fiber, and fructo-oligosaccharides; and wherein the composition is substantially lactose-free and substantially gluten-free. In a related embodiment, the composition of the invention comprises at least 35% rice bran fiber, on a weight percentage basis (i.e., "w/w"). In another related embodiment, the composition of the invention comprises between 35% and 60% w/w rice bran fiber. In yet another related embodiment, the composition of the invention comprises between 35% rice bran fiber and 50% rice bran fiber. In still another related embodiment, the composition of the invention comprises between 35% rice bran fiber and 45% rice bran fiber. In yet another related embodiment, the composition of the invention comprises omega-3 and omega-6 fatty acids. Each of the aforementioned embodiments of the composition of the invention may further comprise phytonutrients and antioxidants.

[0007] In one embodiment of the rice bran composition of the invention, the composition comprises approximately 42% insoluble rice bran fiber, 1.0% soluble rice bran fiber, 21% protein, 13% fat, and 1% available carbohydrates. The invention also provides a composition which comprises the rice bran fiber of Table 5.

[0008] The invention provides a method of reducing the likelihood of cancer in a subject, comprising administering one of the rice bran compositions of the invention to a subject. For example, in one embodiment, the invention provides a method of reducing the likelihood of cancer in a subject comprising administering a rice bran fiber composition to a subject, wherein the composition comprises soluble rice bran fiber, insoluble rice bran fiber, and fructo-oligosaccharides; and wherein the composition is substantially lactose-free and substantially gluten- free; and wherein 35-60% of the composition consists of soluble and insoluble rice bran fiber by weight. In one embodiment of the method, the subject has a higher risk of cancer than an average or normal subject. In a related embodiment, the cancer is selected from the group consisting of cancer of the breast, prostate, lung, colon, rectum,

bladder, pancreas, liver, kidney, skin, mouth, and non-Hodgkin lymphoma. In one embodiment of the method, the cancer is colon cancer.

[0009] The present invention also provides a method for treating metastatic colon cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a rice bran fiber composition to a subject, wherein the composition comprises for example, a soluble rice bran fiber, an insoluble rice bran fiber, and fructo-oligosaccharides; and wherein the composition is substantially lactose- free and substantially gluten- free; and wherein 35- 60% of the composition consists of soluble and insoluble rice bran fiber by weight, where the metastatic colon cancer is treated.

[0010] In another embodiment, the present invention provides a method for treating or preventing a cell proliferative disorder of the colon, comprising administering to a subject in need thereof a therapeutically effective amount of a therapeutically effective amount of a rice bran fiber composition to a subject, wherein the composition comprises for example, a soluble rice bran fiber, an insoluble rice bran fiber, and fructo-oligosaccharides; and wherein the composition is substantially lactose- free and substantially gluten-free; and wherein 35- 60% of the composition consists of soluble and insoluble rice bran fiber by weight, where the cell proliferative disorder of the colon is treated or prevented.

[0011] In yet another embodiment, the present invention also provides a method for inducing cell death in a colon cancer cell, comprising contacting the colon cancer cell with an effective amount of a rice bran fiber composition to a subject, wherein the composition comprises for example, a soluble rice bran fiber, an insoluble rice bran fiber, and fructo- oligosaccharides; and wherein the composition is substantially lactose-free and substantially gluten- free; and wherein 35-60% of the composition consists of soluble and insoluble rice bran fiber by weight, where the contacting induces the cell death in the colon cancer cell.

[0012] In a related embodiment, approximately 10 to approximately 50 grams of the composition are administered to a subject on a daily basis. In yet another related embodiment, the higher risk of cancer in the subject is determined prior to the administration of the rice bran composition. In yet another related embodiment, the determination comprises the use of a genetic test. In yet another related embodiment, the administration of the composition facilitates the apoptosis of cancer cells in the colon.

[0013] In one embodiment, the invention also provides a method of improving gastrointestinal and colon health in a subject, comprising administering a rice bran composition to a

subject. In another embodiment, the invention provides a method of facilitating the growth of bifido bacteria in the intestines, comprising administering a rice bran composition of the invention to a subject. In a related embodiment, the fermentation of the rice bran fiber in the intestines of the subject lowers the pH in the subject's intestines. In another embodiment, the administration of the rice bran fiber leads to an increase in the measured intestinal enzyme activity in the subject. In yet another embodiment, the administration of the rice bran fiber composition of the invention improves the absorption of sodium and water in the intestines.

[0014] In another embodiment, the invention provides a method of decreasing the likelihood of gall stone and kidney stone formation in a subject, comprising administering one of the aforementioned rice bran compositions of the invention to a subject. In yet another embodiment, the administration of the rice bran fiber induces the synthesis of B 12- vitamins in the subject. These and other aspects and embodiments will become more apparent when read with the accompanying figures and detailed description.

BRIEF DESCRIPTION OF THE FIGURES [0015] FIG. 1 illustrates the key differences between flattened, injured or immature absorptive intestinal cells versus tall, healthy, mature absorptive intestinal cells.

[0016] FIG. 2 illustrates the cancer blocking and/or suppressive effects of phytochemicals present in the stabilized rice bran fiber compositions described herein. Specifically, the illustration shows how phytochemicals act at different stages of carcinogenesis.

[0017] FIG. 3 illustrates the steps in the standard Ames assay.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

I. DEFINITIONS

[0018] As used herein the term "stabilized rice bran derivative insolubilized fraction" includes a fraction of stabilized rice bran produced during a partitioning process. Specifically, after the stabilized rice bran aqueous slurry is enzymatically treated as discussed fully below, it is then pumped into a centrifuge where the insoluble fraction precipitates out of the aqueous solution. The insoluble fraction is collected and then dried, and subsequently ground into a powder. This powder is the insoluble portion. In a preferred embodiment, the

constituent parts and their percentages are listed in Table 5. The process for isolating this fraction from rice bran is described in Example 1.

[0019] As used herein the term "stabilized rice bran derivative solubilized fraction" includes a fraction during a partitioning process. Specifically, after the stabilized rice bran aqueous slurry is enzymatically treated, it is then pumped into a centrifuge where the insoluble fraction precipitates out of the aqueous solution. The aqueous material is pumped to a dryer and then dried. This dried aqueous portion produces the soluble fraction.

[0020] As used herein the term "triglyceride" includes a lipid or neutral fat consisting of glycerol combined with three fatty acid molecules.

[0021] In certain aspects, the rice bran fiber of the present invention is a combination of both soluble and insoluble fiber. It has significant amount of fructo-oligosaccharides, the food for bifidobacteria to proliferate in the intestines. Rice bran fiber is associated with potent antioxidants, quality fat and hypoallergenic protein, but is free of lactose and gluten. It is an all-natural derivative of stabilized rice bran (SRB) and is nutritionally superior to any other fiber existing today (see, e.g., Table 4). In one embodiment, the invention provides a rice bran fiber known as RiceMucil . RiceMucil is prepared from SRB following a non- chemical process by which the nutritionally potent fiber is separated from the soluble components into a fine feathery powder (US Patent No. 6,350,473, incorporated by reference herein, in its entirety for all purposes). A detailed description of this embodiment of rice bran fiber is provided in Table 5.

[0022] In another embodiment, the stabilized rice bran fiber prepared according to the above-described process comprises the following ingredients per 10 gram serving: 4.2 grams insoluble fiber; 0.1 grams soluble fiber; 2.1 grams protein; 1.3 grams fat; and 1.0 grams available carbohydrates. The relative amounts of the ingredients are approximate and the amount of each can be changed by approximately 10% without substantially altering the beneficial properties of the fiber.

[0023] Certain stabilized rice bran derivatives are disclosed in the following commonly owned U.S. Patents including: U.S. Patent No. 5,985,344, issued November 16, 1999, entitled, "Process for Obtaining Micronutrient Enriched Rice Bran Oil"; U.S. Patent No. 6,126,943, issued October 3, 2000, and entitled, Method for Treating Hypercholesterolemia, Hyperlipidemia, and Atherosclerosis"; U.S. Patent No. 6,303,586 issued October 16, 2001, and entitled "Supportive Therapy for Diabetes, Hyperglycemia and Hypoglycemia"; U.S.

Patent No. 6,350,473, issued February 26, 2002 and entitled "Method for Controlling Serum Glucose"; U.S. Patent No. 6,558,714, issued May 6, 2003, and entitled "Method for Treating Hypercholesterolemia, Hyperlipidemia, and Atherosclerosis"; U.S. Patent No. 6,733,799 issued May 11, 2004, and entitled "Method for Treating Hypercholesterolemia, Hyperlipidemia, and Atherosclerosis"; and U.S. Patent No. 6,902,739, issued June 7, 2005, and entitled "Method for Treating Joint Inflammation, Pain, and Loss of Mobility." Each of the foregoing patents are hereby incorporated by reference.

II. EMBODIMENTS

[0024] The physiological attributes of dietary fiber depend on their molecular design and solubility in water. When these undigested dietary fibers reach the colon, the bacteria ferment the insoluble fibers, producing short chain fatty acids (i.e., acetic, propionic and butyric acids) and reduce the pH to acidic levels. These acids known as volatile fatty acids are used as an energy source by the mucosal cells of the colonic epithelium. The high viscosity in the gut created by the soluble fiber results in a decreased uptake of glucose and is beneficial to the patient with diabetes. The absorption of lipids through the same mechanism lowers cholesterol.

[0025] Dietary fiber adds to the bulk of the diet by assisting in the transit of food into the gastrointestinal tract. It retains water and softens the stool for easy excretion (see, Normand, F. L. et al, Food Technology 2:90-99 (1987)). Dietary fiber plays an important role in weight reduction as it expands in the stomach occupying space thereby increasing satiety and decreasing appetite. Dietary fiber contains no calories as it is not digested in the human gut. Dietary fiber helps in healthy weight loss as it creates satiety and decrease in appetite.

[0026] Two types of fibers are essential for their functional role in the diet. A combination of soluble and insoluble fibers is required to achieve maximum health benefits. Suggested practices for maintaining a healthy intake of dietary fiber include (1) taking a supplement with a balanced matrix of soluble and insoluble fiber; (2) ingesting 30-35 grams/day total dietary fiber (TDF) which includes 8-10 grams of soluble fiber; and (3) drinking 8 glasses of water a day since fiber supplements absorb water. If an adequate amount of water is taken, the fiber will utilize intestinal fluids resulting in electrolyte imbalance.

[0027] Insoluble fiber consists of celluloses, hemicelluloses and lignins. Lignins are mature cell wall matrices consisting of condensed polyphenols. Whole grains, rice bran, and

wheat bran are rich in insoluble fiber. Mature vegetables, beans, apples and root vegetables are other sources of insoluble fiber. Insoluble fiber passes through the gut unchanged and reaches the colon. Insoluble fiber also helps in maintaining gastrointestinal and colon health (see, Folino, M. et al., J. Nutrition, 125(6):1521-8 (1995)). It aids in the fermentation of undigested food in the colon, binds with the bile salts and bile pigments for excretion, and aids in the proliferation of bifido bacteria by providing an acidic pH.

[0028] During the anaerobic bacterial fermentation of undigested food in the colon, insoluble fiber produces short chain fatty acids (SCFAs) like propionic acid and butyric acid (unlike soluble fibers which produce gases such as carbon dioxide and methane), acidifying the lower luminal pH. This acidic environment in the colon has several beneficial effects such as promoting the growth of friendly bacteria, stimulating cell turn over between the villi and increasing the production of RNA, DNA and protein by the cells. SCFAs also stimulate pancreatic enzyme secretion, increase sodium and water absorption in the colon, and induce the synthesis of B-complex vitamins, especially B 12. Additionally, SCFAs in the colon induce the synthesis of natural killer cells (NF-kB) which are involved in the natural death of cancer cells. This property helps in preventing colon and prostate cancer by gene regulation and apoptosis (cell death of the cancer cells). Insoluble fiber prevents diverticulitis, colon/rectal polyps (Harvard study with 43,881 US male health professionals ranging from 40-75 years), IBS (inflammatory bowel syndrome), ulcerative colitis and cancer. A condition consisting of inflamed colon may arise from food allergies, stress or bacterial/viral infections.

[0029] Soluble fiber is soluble in water, forming a highly viscous gel-like fluid with food in the small intestines. The high viscosity is responsible for the delayed absorption of glucose in the intestines and reduces the post prandial glucose levels in the diabetics. This high viscosity also helps in the reduction of the serum cholesterol and triglyceride levels and increasing HDL levels (see, Madar, Z., Am. J. Clin. Nutr., 38:388 (1983)). Fibers, both soluble and insoluble, prevent the re-absorption of the bile acids from the small intestines back into circulation thereby reducing the circulating cholesterol. Fibers also dissolve the gallstones in the gallbladder.

[0030] Examples of soluble fibers are gums, pectins, mucillages, galactomannans, arabinogalactans, beta glucans, from barley and non-starchy water-soluble polysaccharides containing hemicelluloses. Most fruits, vegetables, barley, psyllium and oat bran have soluble fibers. Soluble fiber such as pectin is composed of repeated units of galactouronic

acids that have been methylated and have β 1-4 covalent linkages. Gums and mucillages are also soluble fibers composed of hexose and pentose units with β 1-4 covalent linkages.

[0031] In addition to 42% fiber, the RiceMucil™ embodiment of the present invention contains proteins, antioxidants and phytonutrients, such as gamma oryzanol, inositol hexaphosphate (IP6), and phytosterols, which provide excellent nutrition to the gut and help to maintain a healthy and clean gut. It also contains a short chain sugar complex, known as fructo-oligosaccaride (FOS), which is not digestible in the gut and serves as food to the friendly bacteria, such as Lactobacillus acidophilus or Lactobacillus sporogens.

[0032] These friendly bacteria aid in the fermentation of the undigested food around the intestinal villi, producing short chain fatty acids, such as butyric and propionic acids in the gut. Scientific data indicates that the short chain fatty acids play an important role in the prevention of colon cancer. Other commercially available dietary fibers produce carbon dioxide and methane gases during colonic fermentation, resulting in abdominal distention and discomfort.

[0033] RiceMucil in the diet prevents constipation and irregular bowel movement. It provides bulk to the waste and decreases the colonic transit time by several-fold, thereby reducing the risk of colorectal cancer.

[0034] Cancer is a result of free radical damage to DNA and protein in various organs of the body. Cancer formation occurs in three stages: (1) an initiation stage, (2) a latent stage of progression and (3) tumor formation stage. Certain natural products are known to inhibit cancer by two major mechanisms. Specifically, the products may act as blocking agents and/or as suppressing agents {see, Figure 2). By blocking attacks on DNA and protein by active carcinogens, cancer can be prevented at the initiation stage itself. Even after the formation of DNA adducts, cancer can be arrested during the latent period by the suppressor mechanism, preventing the proliferation of diseased cells.

[0035] Components of the rice bran fiber compositions described herein are involved in the above-mentioned blocking and suppressing mechanisms. For instance, ferulic acid increases liver microsomal phase 2 detoxifying enzymes several fold and inhibits phase 1 carcinogen activating enzymes (Manorama, 1993). In the presence of sufficient amounts of ferulic acid and similar compounds, a carcinogen entering the body cannot reach the DNA because it is not activated to a reactive metabolite. The body's detoxification mechanisms can eliminate

the carcinogen before it is capable of doing harm. Thus, this blocking mechanism prevents cancer formation.

[0036] In contrast, as described above, the suppressor mechanism acts on previously formed DNA adducts to suppress the progression of cancer. For example, the signal transduction pathway created by T3, IP6 and other phytonutrients, appears to switch the cell programming from a proliferative to a differentiation pathway, which can reverse cancer progression. IP6 has been shown to act both as a blocking agent and as a suppressing agent. Data on breast cancer, lung cancer, and colon cancer, as well as the cancer of several organs (Shamsuddin, 1995), shows a strong chemopreventive effect of IP6 treatment, where IP6 appears to function both as a blocking agent and as a suppressive agent.

[0037] Rice bran polyphenols also have a chemopreventive effect (Hudson et al., 2000). Tocotrienols are another group of rice bran phytochemicals having a chemopreventive effect which have been shown to inhibit breast cancer (see, Nesaratnam et al., 1997). The polysaccharides of rice bran include α-glucan, the anti-tumor effect of which was demonstrated by the inhibition of gastrointestinal carcinogenesis (see, Akeshita, 1992). Rice bran agglutinin was shown to induce apoptosis of cancer cells by the mechanism of cell cycle dysregulation (see, Myoshi et al., 2001). In addition to these compounds, rice bran includes potent antioxidants that prevent the free radical attack at several stages of carcinogenesis. Some of these phytochemicals (e.g., phytosterols) get incorporated into cell membranes and improve the cell integrity and fluidity of the cell membrane. This prevents the carcinogens from entering the cell. An anti-tumor polysaccharide from rice bran has been isolated (see, Ito et al. 1985) and has been demonstrated to have anticancer and immune enhancing properties (see, Nakumura, 1992). Another anti -tumor lipoprotein fraction of rice bran has been shown to induce apoptosis and inhibit the growth of cultured human endometrial adenocarcinoma cells (see, Fan et al, 2000).

[0038] In addition to their cancer preventative effects, the rice bran fiber compositions of the present invention are useful for lowering cholesterol and treating hyperlipidemia. RiceMucil ^™ was tested for RiceMucil ^™ its cholesterol lowering effect in an eight-week human study. A total of 20 individuals with clinically established Type 1 diabetes, and 26 individuals with Type-2 diabetes, with lipid abnormalities, were treated with RiceMucil ^™ by administering in two equally divided doses of 10 grams each, one taken before breakfast and another before dinner in milk/juice for a period of eight weeks. RiceMucil ^™ produced

significant reduction in total cholesterol, LDL cholesterol, apo B, and triglycerides in both Type 1 and Type 2 diabetics with percentage reductions ranging between 7-18%. There was no change in HDL cholesterol. Results of the above study indicate clearly that RiceMucil ^™ controls hyperlipidemia very effectively (see, Cheruvanky et al., U.S. Patent No. 6,126,943).

[0039] RiceMucil and other embodiments of the rice bran fiber of the present invention reduces blood cholesterol by premature emptying of the gall bladder and trapping the bile salts, which thereby prevent re-absorption. It is believed that the high quality fiber in the compositions disclosed herein, along with the major bioactive compounds (such as tocopherols, tocotrienols, gamma oryzanol, phytosterols, and inositol) present in the product, synergistically help in the management of heart diseases.

[0040] A diet rich in fiber also helps to prevent gallstone formation. This is due to improved cholesterol levels, which prevent high cholesterol deposits (in crystallized form) from lodging in the gall bladder. Researchers have shown that the effectiveness of a high rice bran dietary fiber intake in reducing renal calcium excretion (see, Jahnen, A. et al., Urol. Res. 20:3-6 (1992)). A highly nutritious dietary fiber complex derived from rice bran, such as

RiceMucil , helps in preventing gall stone formation by keeping the cholesterol levels under check, as well as by effectively reducing renal calcium excretion, which are the two main causes of gallstones.

[0041] RiceMucil helps to stabilize blood sugar levels in Type 1 and Type 2 diabetics because it contains the right type of fiber, which can slow the rate of sugar absorption into the blood stream, thereby reducing the level of insulin required to process the food at any given moment.

[0042] In another embodiment, the stabilized rice bran fiber compositions of the present invention stimulate microvilli in the intestine and extend their age (see, FIG. 1). Other fibrous compositions and diets can erode microvilli and inhibit the ability of microvilli to synthesize and maintain active disaccharidases, either directly or by facilitating the invasion of enteroadherent bacterial pathogens (see, e.g., Fleckenstein and Kopecko, J Clin Invest, 107:27-30). The presence of eroded or damaged microvilli thus impairs the body's ability to digest disaccharides.

[0043] Constipation and irregularity of bowel movements are also major consequences of fiber-deficient diets. Ingesting the correct amounts and types of fibers increases the fecal

bulk while also absorbing water into the large intestine. This creates a softer stool that exerts less pressure on the colon walls and is more easily excreted. RiceMucil contains 42% insoluble fiber, and is an ideal help for constipation. It increases fecal bulk and reduces the length of time of gastric emptying, thereby giving instant relief to people suffering from constipation.

[0044] Fiber is preferably taken between meals or on an empty stomach in the morning and evening. Fiber contains phytates and these tend to absorb minerals such as calcium, magnesium, manganese, and iron. Consumption of fiber is therefore most effective when taken on an empty stomach.

[0045] Twenty grams of RiceMucil ^™ a day, when given in two equal doses of 10 grams each before breakfast and before dinner to Type 1 and Type 2 diabetic patients for eight weeks, significantly reduced glucose parameters (see, Cheruvanky et al, U.S. Patent No. 6,303,586 Bl).

[0046] RiceMucil ^™ taken half an hour before mealtime or with meals, forms the bulk of food replacing high-calorie diets. A high fiber, low fat, low calorie meal is recommended for weight control. RiceMucil ^™ , which has 20% protein and significant quantities of gamma oryzanol and several other phytonutrients, is an effective diet for weight management.

[0047] RiceMucil is all natural, non-genetically engineered, chemical-free, and manufactured according to the strict GMP conditions of FDA. Every batch is tested for quality and safety in the laboratory.

[0048] Because of the antimutagenic properties of the rice bran-derived composition of the invention, physicians who have diagnosed a patient as having a form of cancer, e.g., colon cancer, may recommend that the patient be treated with (e.g., consume) one or more of the rice bran-derived compositions of the present invention. A physician may also recommend that patients who are at higher risk of a cancer, e.g., colon cancer, also be treated with (e.g., consume) one or more of the rice-bran derived compositions of the present invention. A patient with a higher risk of cancer is a patient who is more likely than an average member of the population to develop any particular form of cancer, i.e., the risk could be the risk of a future diagnosis of a specific cancer such as colon cancer or lung cancer, or the risk of a future diagnosis of cancer generally. Patients at higher risk of cancer include, without limitation, patients who smoke, patients who are genetically more susceptible to cancer,

patients who work in environments where they are exposed to a higher concentration of mutagenic substances than patients in the general population, etc.

[0049] In a preferred aspect, compositions of the present invention may be used to treat colon cancer or cell proliferative disorders of the colon. In one aspect, colon cancer includes all forms of cancer of the colon. In another aspect, a colon cancer to be treated includes carcinoma, sarcoma, and adenocarcinoma. In another aspect, colon cancer includes sporadic and hereditary colon cancers. In another aspect, colon cancer includes malignant colon neoplasms, carcinoma in situ, leiomyosarcomas, typical carcinoid tumors, and atypical carcinoid tumors. In another aspect, colon cancer includes adenocarcinoma, squamous cell carcinoma, signet ring cell adenocarcinoma and adenosquamous cell carcinoma. In another aspect, colon cancer is associated with a hereditary syndrome selected from the group consisting of hereditary nonpolyposis colorectal cancer (HNPCC), familial adenomatous polyposis (FAP),Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndrome and juvenile polyposis. In another aspect, colon cancer is caused by a hereditary syndrome selected from the group consisting of hereditary nonpolyposis colorectal cancer (HNPCC), familial adenomatous polyposis (FAP), Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndrome and juvenile polyposis.

[0050] In certain aspects, the colon cancer that is to be treated has been staged according to the American Joint Committee on Cancer (AJCC) TNM classification system, where the tumor (T) has been assigned a stage of Tx, Tis, Tl, T2, T3, T4; and where the regional lymph nodes (N) have been assigned a stage of NX, NO, Nl, N2, N2a, N2b, N3, N3a, N3b, or N3c; and where distant metastasis (M) has been assigned a stage of MX, MO, or Ml. In another aspect, a colon cancer that is to be treated has been staged according to an American Joint Committee on Cancer (AJCC) classification as Stage 0, 1, II, HA, HB, III, IIIA, IIIB, HIC and IV colon cancer. In another aspect, a colon cancer that is to be treated has been assigned a grade according to an AJCC classification as Grade GX {e.g., grade cannot be assessed), Grade 1 , Grade 2, Grade 3 or Grade 4. In another aspect, a colon cancer that is to be treated has been assigned a grade according to the Dukes staging system of A, B, or C. In another aspect, a colon cancer that is to be treated has been assigned a grade according to the Astler- Coller staging system of A, B 1, B2, B3 or Cl, C2, C3, or D.

[0051] In summary, the present invention provides rice bran fiber compositions which are hypoallergenic, lactose-free and gluten- free, as well as methods of administering those

compositions for the purpose of achieving bowel regularity and relieving constipation; normalizing blood glucose levels; normalizing serum cholesterol and lipid levels; reducing the risk of coronary heart disease; achieving healthy weight loss; preventing cancer of the colon; improving gastrointestinal and colon health; improving the colonization of probiotics (friendly bacterial colonies) in the intestines due to a significant amount of fructooligosaccharides (FOS); improving sodium and water absorption in the colon; improving the intestinal enzyme production due to the low pH created by short chain fatty acids (SFA); preventing gallstone and kidney stone formation; and inducing the synthesis of B-Complex vitamins.

III. EXAMPLES

EXAMPLE 1

[0052] This example illustrates the preparation of rice bran derivatives.

[0053] In order to generate the rice bran derivatives for use in the present invention, the rice bran is first stabilized, and then it is further separated into at least two fractions. These include, but are not limited to, a stabilized rice bran soluble derivative and a stabilized rice bran insoluble derivative. Preferably, the separation into the rice bran derivatives includes a nonchemical process i.e., an enzymatic process. In this process, partitioning or fractionation preferably proceeds as outlined hereinafter.

[0054] The stabilized rice bran is made into about a 15% to about 35% slurry, preferably, a 20-25% slurry with potable water. An enzyme, which can include, but is not limited to, a dextranase, a maltase, α-amylase, and various other carbohydrate cleaving enzymes, is added to the batch converting the starch to dextrins. The slurry is heated to about 150° F to about 200° F using, for instance, a steam injection cooker, a heat exchanger, or other heating method. The slurry is then pumped to a horizontal centrifuge wherein the insoluble fraction is separated. The insoluble fraction is collected and then dried on a belt dryer, and subsequently ground into a powder. This powder is the stabilized rice bran insoluble fraction. The aqueous material is pumped to a drum dryer and then dried. This dried aqueous portion produces the stabilized rice bran solubilized fraction.

[0055] The enzyme treated stabilized rice bran can be generated using the rice bran slurry as described above. As such, in another aspect, the present invention relates to the process for making an enzyme treated stabilized rice bran derivative, comprising: admixing stabilized

rice bran with an aqueous solution to form about a 15% to about a 35% aqueous rice bran slurry, preferably a 20% to about a 30% aqueous rice bran slurry w/w; adding an enzyme to the aqueous rice bran slurry to convert starch to dextrin, thereby forming an enzyme treated slurry and then directly drying the enzyme treated slurry to form an enzyme treated stabilized rice bran derivative.

[0056] In a preferred embodiment of the foregoing process, after the enzyme is added to the slurry, the slurry is heated to about 100° F. to about 200° F. Preferably, the slurry is heated to about 150° F to about 200° F. The slurry is then dried, wherein the drying is accomplished by a process such as belt drying, spray drying, drum drying and air drying. The drum drying process is preferred.

[0057] These stabilized rice bran derivatives are also available commercially from the NutraCea company of El Dorado Hills, California. Specifically, the insoluble derivative of stabilized rice bran is available as RiceMucil Fiber Complex and the soluble derivative is available as RiSolubles ^® .

[0058] The stabilized rice bran derivatives can take a variety of forms. They can be a powder, a food, a food supplement, a medical food, a liquid, a beverage, an emulsion or mixture thereof. In addition, they can be incorporated into other edible materials. To incorporate the rice bran derivative into the diet of a mammal various options include, but are not limited to, simply sprinkling the derivative on another food substance (i.e., salad, bread, cereal, etc.) being a major ingredient in a multigrain ready to eat cereal, incorporating it into a baked product (breads, muffins, waffles, etc), pasta, healthy dessert and snacks (athletic bar, healthy drink, etc.) and high fiber foods.

[0059] Stabilized rice bran contains about 18-23% fat, about 23-35% dietary fiber, about 12-16% protein, about 8-36% total carbohydrate and many potent microcomponents. Rice bran solubles contains about 15-40% fat, preferably 23-30% fat; about 0% to 25% dietary fiber, preferably about 0-20% dietary fiber; about 0% to 15% protein, preferably 6-9% protein and 25% to about 80% carbohydrates, preferably about 27-66% simple carbohydrate and is a water soluble fraction. Stabilized rice bran insoluble derivative contains about 5%- 20% fat, preferably 11-16% fat; about 40-65% dietary fiber, preferably 40-60% dietary fiber, and about 10-30% protein, preferably 18-22% protein (see Table 5).

EXAMPLE 2

[0060] This Example describes tests used to demonstrate the antimutagenic properties of RJceMucil ^™ and its components.

A. Overview of Salmonella mutagenicity test.

[0061] The Salmonella mutagenicity test, or bacterial reverse mutation assay, is also commonly known as the Ames test (after Dr. Bruce Ames, who developed the test with is colleagues in UC Berkeley in the 1970s). The principle of the test is to expose histidine- dependent Salmonella typhimurium strains (the tester strains, which have artificially induced point mutations) to a compound to be examined in a histidine (His) deficient medium. His- independent bacterial colonies may arise from spontaneous reversions (backward mutations) or chemically induced reversions.

[0062] The Ames test is a screening assay for carcinogens that uses bacteria to detect chemical mutagens. It is based on the premise that most carcinogens induce cancer because they are mutagens. If these agents are shown to be mutagenic for bacteria, they may also alter DNA in eukaryotic cells. In the Ames test, a strain of Salmonella typhimurium auxotrophic for histidine (his-), defective in dark repair of mutations (uvrB), and unable to synthesize a portion of the cell wall (rfa) is exposed to chemicals. The rate of reversion (back mutation) to prototrophy caused by the chemical is measured.

[0063] The test is performed by spreading a suspension of the Salmonella in soft agar over a layer of minimal medium. The suspected mutagen may be added to the soft agar along with the Salmonella or it may be placed on a disk in the center of the seeded layer after it is spread. The soft agar contains an amount of histidine that supports only a few rounds of bacterial cell division. This is essential because many mutagens work only on replicating DNA. If a back mutation to prototrophy occurs, visible colonies develop. The reversion rate is compared to a control plate (no chemicals added) and is proportional to the mutagenicity of the chemical. Many chemicals that are non-mutagenic by the Ames test can induce tumors. Some of these chemicals are metabolized to a mutagenic form in the liver. To mimic the activation process, the Ames test usually contains an extract of mammalian liver enzymes.

[0064] In a standard Ames assay a rat liver homogenate is used to provide the cytochromes P450 necessary for activation of mutagens to their reactive forms. The suspected mutagen is mixed with the homogenate, where it is metabolized to its reactive form, and then an

appropriate tester strain of Salmonella typhimurium is added to the mix. The activated mutagen enters the bacterium and reacts with DNA to cause mutations; mutations in the operon for histidine biosynthesis that restore function ("reversions") allow the bacterium to grow on histidine-defϊcient medium, and result in visible colonies on the assay plate. The greater the number of colonies, the more mutagenic the chemical.

[0065] The following bacterial strains are used in this assay: TA98 (frame-shift mutation in histidine gene); TAlOO (base-pair substitution in histidine gene); TAl 535 (base-pair substitution in histidine gene); TA1537 (frame-shift mutation in histidine gene); and TA102 (base-pair substitution in histidine gene). The two standard testing strains (TAl 535 and TA1537) are used in combination with the strains, TA98, TAlOO and TA102, which include the plasmid pKMIOl, as well as their particular histidine gene mutation. The plasmid- carrying derivatives (TA98, TAlOO and TA 102) have an increased sensitivity to certain mutagens because pKMIOl codes for an error-prone DNA repair system. Strain TA 102 has its histidine gene mutation located on a multi-copy plasmid, pAQl.

[0066] Many chemicals are not mutagenic (or carcinogenic) in their native forms, but they are converted into mutagenic substances by metabolism in the liver. Since the Salmonella bacterium does not have the same metabolic capabilities as mammals, some test protocols utilize extracts of rat or hamster liver enzymes (S9) to promote metabolic conversion of the test chemical. This permits the investigator to determine if a chemical must be metabolized to express mutagenic activity. Some mutagenic chemicals are active with and without metabolism, while others are active only under one condition or the other.

[0067] In the Salmonella assay, a test tube containing a suspension of one strain of Salmonella typhimurium plus S9 mix or plain buffer without S9, is incubated for 20 minutes at 37° C with the test chemical. Control cultures, with all the same ingredients except the test chemical, are also incubated. In addition, positive control cultures are also prepared; these contain the particular bacterial tester strain under investigation, the various culture ingredients, and a known potent mutagen. After 20 minutes, agar is added to the cultures and the contents of the tubes are thoroughly mixed and poured onto the surface of petri dishes containing standard bacterial culture medium. The plates are incubated, and bacterial colonies that do not require an excess of supplemental histidine appear and grow. These colonies are comprised of Salmonella that have undergone reverse mutation to restore

function of the histidine-manufacturing gene. The number of colonies is counted after 2 • days.

B. Mutagenicity testing of rice bran oil unsaponifϊable fraction.

[0068] We have carried out three experiments with rice bran oil and its unsaponifiables using a Salmonella typhimurium bacterial assay system.

[0069] Rice bran oil unsaponifiable extracts were prepared and separated into polar and non-polar fractions according to Taylor et al. {Journal of the American Oil Chemists Society, (1983) 60:576). Rice bran has 20% fat. When the fat is extracted from rice bran or rice bran fiber, the fat is known as rice bran oil. Rice bran oil concentrates all the fat-soluble phyto nutrients and antioxidants of rice bran into the oil. The non-glyceride portion of the oil is known as the unsaponifϊable fraction, containing concentrated rice bran phytonutrients and antioxidants of the rice bran. This unsaponofiable fraction is further separated into polar and non-polar fractions, per the protocols described in Taylor et al. (1983).

[0070] Assays were conducted using Salmonella typhimurium strains TA 98 and TA 100, plated at a density of 6.6 x 10 ⁹ bacteria per plate. The rat liver microsomal S9 extracts were prepared according to Maron and Ames (Mutat Res., (1983) 113: 173-215). Reported values are the mean of four plates. The mutagens benzo(a)pyrene (5 μg/plate) and sodium azide (1 μg/plate) were used as positive controls for TA 98 and TA 100 respectively. DMSO extracts of the rice bran oil fractions (0.2 ml) was mixed with 0.5 ml of S9 mixture and 0.1 ml of overnight grown bacterial culture. The mixture was then poured on minimal, glucose agar medium plates and incubated in the dark at 37 ⁰ C. After 48 hours, the number of colonies of histidine prototrophs on the plates were counted.

C. Results

Table-1 : Mutagenicity test in Salmonella typhimurium TA 98 strain

[0071] Each of the above values represents mean number of revertants from 4 plates with their SD. No significant difference was observed between the rice bran unsaponifiables and peanut oil unsaponifiables. The TA 98 control without benzo(a)pyrene yielded 24 + 4 colonies; the TA 98 control with benzo(a)pyrene yielded 58 + 2 colonies. These data show that the constituents of rice bran unsaponifiables are non-mutagenic.

Table-2: Mutagenicity test in Salmonella typhimurium TA 100 strain

[0072] Each of the above values represents mean number of revertants from 4 plates with their SD. No significant difference was observed between the rice bran unsaponifiables and peanut oil unsaponifiables. The TA 100 control without azide yielded 158 + 14 colonies; the TA 100 strain treated with azide yielded 485 + 18 colonies. These data show that the constituents of rice bran unsaponifiables are non-mutagenic.

Table-3: Mutagenicity of Rice Bran Oil before and after 6 hours heating at 18O ⁰ C

[0073] The above results demonstrated that neither the polar or non-polar fractions exhibited mutagenicity towards the His- strains, regardless of the presence of metabolic activators. Although the formation of mutagens are associated with heating and cooking conditions, rice bran oil is not detectably affected by such conditions. Taken together, the results and discussion above show that rice bran and its product.

EXAMPLE 3

[0074] This example illustrates a study to analyze the effect of rice bran derivatives on carcinogen-induced colonic aberrant crypt foci (ACF) formation in rat.

A. Experimental

[0075] Aberrant crypt foci (ACF) are recognized as early pre-neoplastic lesions in the colon, and have consistently been observed in experimentally induced colon carcinogenesis in laboratory animals (see, McLellan, E. A. et al, Cancer Res., 51, 5270-5274, (1991) and Wargovich, M. H., et al, Cancer Epidemiol Biomarkers & Prev., 5, 355-360, (1996)). It has also been shown that these lesions are present in the colonic mucosa of patients with colon cancer and have suggested that aberrant crypts are putative precursor lesions from which adenomas and carcinomas may develop in the colon (see, Pretlow, T. P., et al., J. Cell. Biochem., 16G (Suppl.), 55-62, (1992)). It is known that ACF express mutations in the ape gene and ras oncogene that appear to be biomarkers of colon cancer development (see, Vivona, A. A., et al., Carcinogenesis (Lond.) 14, 1777-1781, (1993)). There is some evidence that several inhibitors of ACF formation reduce the incidence of colon tumors in laboratory animals suggesting that ACF induction can be used to evaluate novel agents for their potential chemopreventive properties against colon cancer. [0076] In this example, Weanling male rats are obtained from Charles River Breeding Laboratories (Kingston, N. Y., USA). All experimental rice bran ingredients are obtained from Nutracea, El Dorado Hills, CA. The experimental rats are in quarantine for 1 week and are randomly distributed by weight into various dietary groups and are transferred to an animal holding room where they are housed in plastic cages, three rats per cage, under controlled conditions of a 12 h light/ 12 h dark cycle, 50% relative humidity, and their diets are strictly controlled.

[0077] Beginning at 5 weeks of age, groups of animals are fed either a control of rat chow, or alternatively, experimental diets of stabilized rice bran. All animals except the vehicle- treated rats receive azomethane (AOM) s.c. once weekly at 7 and 8 weeks of age at a dose rate of 15 mg/kg body weight/week. There are three groups of animals i.e., 1) Controlled diet (rat chow) and AOM treated; 2) experimental diet of rice bran and AOM treated; 3) Control diet of rat chow and saline (vehicle).

[0078] Azomethane is known to induce aberrant crypt foci. Animals intended for vehicle treatment are given an equal volume of normal saline. The rats on control rat chow or experimental rice bran diets are given AOM until the termination of the study, when they are 16 weeks of age.

[0079] All animals are sacrificed by CO ₂ euthanasia. The colons are removed, flushed with Krebs-Ringer solution, opened from cecum to anus, and fixed flat between two pieces of

filter paper in 10% buffered formalin. After a minimum of 24 h in buffered formalin, the colons are cut into 2-cm segments, placed in a Petri dish containing 0.2% methylene blue in Krebs-Ringer solution and kept for 5-10 min. The colons are placed, mucosal side up, on a microscope slide and observed through a light microscope. The ACF are recorded according to standard procedures. Aberrant crypts are distinguished from the surrounding normal crypts by their increased size, significantly increased distance from lamina to basal surface of cells, and the easily discernible pericryptal zone. Crypt multiplicity are determined as the number of crypts in each focus and categorized as those containing up to three, or four or more aberrant crypts/focus. All colons are scored by one observer without knowing the identity of agents under study; scores are checked at random by a second observer.

B. Results

[0080] The body weights of AOM and vehicle-treated animals fed the control diets and experimental diets containing rice bran derivatives are comparable. There are no signs of any adverse effects in liver, kidney, stomach, intestine or lungs of animals fed rice bran.

[0081] The animals administered saline (vehicle) and fed the control and experimentals diets containing rice bran show no evidence of ACF formation in the colon. In the animals fed the control diet, AOM treatment induces about 120 ACF/colon. ACF are predominant in the distal colons. Efficacy end points in this study are inhibition of the total number of ACF/colon, as well as reducing the number of multi crypt clusters (2 or more) of aberrant crypts/focus.

[0082] Administration of rice bran in the diet significantly suppresses the total number of ACF/colon as compared to the control diet; the degree of inhibition is significant. Crypt multiplicity in terms of 2 or 3 aberrant crypts/focus is inhibited in animals fed rice bran. [0083] Because multiplicity of aberrant crypts has been a probable predictor of colon tumor outcome (see, Pretlow, T. P., et al, Carcinogenesis (Lond.), 13. 1509-1512, (1992)), the present study uses this criterion to evaluate rice bran diet for potential inhibitory properties. [0084] The results of the present study indicate that orally taken rice bran inhibits AOM- induced colonic ACF formation in rats supporting the potential colon tumor inhibitory properties of rice bran. The experiment demonstrates the preventive and ACF inhibitory properties of rice bran.

TABLE 4

COMPARISON OF HEALTH BENEFITS AMONG FIBERS AVAILABLE IN THE MARKET

TABLE 5

RICEMUCIL ^™

INGREDIENTS

A non-chemically modified water-insoluble fraction of stabilized rice bran. It contains 4 grams of insoluble fiber, several minerals, EP6, antioxidants, and 2 grams of hypoallergenic protein (per serving).

GUARANTEED SPECIFICATIONS

Protein 18-22%

Fat 11-16%

Total Carbohydrates 50-60%

Total Dietary Fiber 40-50%

Soluble Fiber 0-1%

Ash 8-12%

Moisture 1-5%

Free Fatty Acids < 4%

MICROBIOLOGICAL

Microbiologically safe and pure.

PHYSICAL

Appearance light, fibrous, solid

Color brown/tan

Flavor bland

Bulk Density (g/cc) 0.26

TYPICAL ANALYTICAL DATA

MACRONUTRIENTS (g/lOOg)

Protein 20.50

Fat 13.50

Saturated Fatty Acid 2.40

Total Carbohydrate 52.50

Available Carbohydrate 10.50

Ash 10.00

Moisture (100 degree vac.) 3.50

Crude Fiber 12.20

Total Dietary Fiber 42.00

Soluble Fiber 0.78

Caloπes/lOOg. 245.50

TABLE 5 (cont.)

VITAMINS

Vitamin A, Carotenoids (mcg/lOOg) oCarotene

0-Carotene

Lycopene 2 60

Lutein 0 00

Zeaxanthrn 0.13

Precryptoxanthin/Cryptoxanthin 24 80 ή O. A HCU\

Total Carotenoids 0 60

Vitamin B Complex (mg/100g) 34 53

Vitamin B 1

Vitamin B2 2 00

Vitamin B 3 0.19

Vitamin B5 30.60

Vitamin B 6 1 90

Vitamin B 12 (mcg/100g) 1.70

Vitamin C(mg/100g) < 0 500

Vitamin E Complex (mg/lOOg) < 0 500

Tocopherols (T)

Tocotπenols 1 20

2.50

Total Tocols 3 70

Other Micronutrients (mg/100g)

Folic Acid < 25.00

Bio tin 11.04

Choline 70.50

Inositol 1314 00

Gamma-Oryzanol 174.10

Phytosterols (mg/100g)

Beta-Sitosterol 146.46

Stigmasterol 67.15

Campesterol 90.40

Brassicasterol 13.20

317.21

Total Phytosterols

MINERALS (mg/100g)

Sodium 16.00

Potassium 1670.00

Calcium 92.50

Magnesium 1223.30

Phosphorous 2330.00

Manganese 38.40

Iron 11.60

Copper 0.09

Zinc 9.34

Chromium (ppm) < lppm

Total Sugars(gflOOg) 4 ?ή

(No Lactose)

[0085] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification in their entirety for all purposes.

[0086] Although the invention has been described with reference to preferred embodiments and examples thereof, the scope of the present invention is not limited only to those described embodiments. As will be apparent to persons skilled in the art, modifications and adaptations to the above-described invention can be made without departing from the spirit and scope of the invention, which is defined and circumscribed by the appended claims.