US20100197594A1 - Methods for using soy peptides to inhibit h3 acetylation, reduce expression of hmg-coa reductase and increase ldl receptor and sp1 expression in a mammal - Google Patents

Methods for using soy peptides to inhibit h3 acetylation, reduce expression of hmg-coa reductase and increase ldl receptor and sp1 expression in a mammal Download PDF

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US20100197594A1
US20100197594A1 US12/756,126 US75612610A US2010197594A1 US 20100197594 A1 US20100197594 A1 US 20100197594A1 US 75612610 A US75612610 A US 75612610A US 2010197594 A1 US2010197594 A1 US 2010197594A1
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lunasin
expression
compound
composition
acetylation
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Alfredo Flores Galvez
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SL Technology Inc
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Priority claimed from US11/532,528 external-priority patent/US7731995B2/en
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Assigned to SL TECHNOLOGY, INC. reassignment SL TECHNOLOGY, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SOY LABS, LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/05—Mashed or comminuted pulses or legumes; Products made therefrom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/005—Enzyme inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • This disclosure relates generally to a class of peptides that provide mammals with a variety of health related benefits. More specifically, the present disclosure related to using soy peptides to inhibit H3 acetylation, reduce expression of HMG-CoA reductase, increase LDL receptor and Sp1 expression, protect against or prevent the expression of Matrix metalloproteinase (MMP-1), the breakdown of collagen, photoaging and the formation of skin wrinkles.
  • MMP-1 Matrix metalloproteinase
  • H3 acetylation, expression of HMG-CoA reductase and LDL receptor and Sp1 expression in mammals pays a significant role in various health related factors, including but not limited to total and cholesterol levels, cancer prevention, and UV related skin damage. Accordingly, manipulation and control of these biological mechanisms or factors would provide numerous health related benefits and provide researches with new avenues to develop new therapies.
  • biological activity and “bioactivity” refer to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition, or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures. Biological activities may be observed and measured in in vitro systems designed to test or use such activities also.
  • biologically active refers to a molecule having structural, regulatory and or biochemical functions of a naturally occurring lunasin molecule.
  • a “combination” refers to any association between two or among more items.
  • disease and “disorder” are used interchangeably to describe a state, signs, and/or symptoms that are associated with any impairment of the normal state of a living animal or of any of its organs or tissues that interrupts or modifies the performance of normal functions, and may be a response to environmental factors (such as malnutrition, industrial hazards, or climate), to specific infective agents (such as worms, bacteria, or viruses), to inherent defect of the organism (such as various genetic anomalies, or to combinations of these and other factors.
  • environmental factors such as malnutrition, industrial hazards, or climate
  • specific infective agents such as worms, bacteria, or viruses
  • inherent defect of the organism such as various genetic anomalies, or to combinations of these and other factors.
  • an effective amount refers to the amount of a composition (e.g., comprising Lunasin) sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
  • administering refers to the act of giving a drug, pro-drug, or other agent, or therapeutic treatment (e.g., compositions of the present invention) to a subject (e.g., a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs) and/or to direct, instruct, or advise the use of the composition for any purpose (preferably, for a purpose described herein).
  • a subject e.g., a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs
  • direct, instruct, or advise the use of the composition for any purpose (preferably, for a purpose described herein).
  • administration of one or more of the present compositions is directed, instructed or advised, such direction may be that which instructs and/or informs the user that use of the composition may and/or will provide one or more of the benefits described herein.
  • Exemplary routes of administration to the human body can be through the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, vaginal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.
  • injection e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.
  • Administration which is directed may comprise, for example, oral direction (e.g., through oral instruction from, for example, a physician, health professional, sales professional or organization, and/or radio or television media (i.e., advertisement) or written direction (e.g., through written direction from, for example, a physician or other health professional (e.g., scripts), sales professional or organization (e.g., through, for example, marketing brochures, pamphlets, or other instructive paraphernalia), written media (e.g., internet, electronic mail, or other computer-related media), and/or packaging associated with the composition (e.g., a label present on a package containing the composition).
  • written includes through words, pictures, symbols, and/or other visible descriptors. Such direction need not utilize the actual words used herein, but rather use of words, pictures, symbols, and the like conveying the same or similar meaning are contemplated within the scope of this invention.
  • co-administration refers to the administration of at least two agent(s) (e.g., composition comprising Lunasin and one or more other agents—e.g., a protease enzyme inhibitor) or therapies to a subject.
  • agent(s) e.g., composition comprising Lunasin and one or more other agents—e.g., a protease enzyme inhibitor
  • the co-administration of two or more agents or therapies is concurrent.
  • a first agent/therapy is administered prior to a second agent/therapy.
  • formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art.
  • agents or therapies when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone.
  • co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and/or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.
  • treatment encompasses the prevention, improvement and/or reversal of the symptoms of disease (e.g., skin aging).
  • a composition which prevents or causes an improvement in any parameter associated with disease may thereby be identified as a therapeutic composition.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures.
  • those who may benefit from treatment with compositions and methods of the present invention include those already with a disease and/or disorder (e.g., elevated cholesterol levels) as well as those in which a disease and/or disorder is to be prevented (e.g., using a prophylactic treatment of the present invention).
  • the term “at risk for disease” refers to a subject (e.g., a human) that is predisposed to experiencing a particular disease.
  • This predisposition may be genetic (e.g., a particular genetic tendency to experience the disease, such as heritable disorders), or due to other factors (e.g., age, weight, environmental conditions, exposures to detrimental compounds present in the environment, etc.).
  • factors e.g., age, weight, environmental conditions, exposures to detrimental compounds present in the environment, etc.
  • the terms “individual,” “host,” “subject” and “patient” refer to any animal, including but not limited to, human and non-human animals (for example, without limitation, primates, dogs, cats, cows, horses, sheep, rodents, poultry, fish, crustaceans, etc.) that is studied, analyzed, tested, diagnosed or treated.
  • the terms “individual,” “host,” “subject” and “patient” are used interchangeably, unless indicated otherwise.
  • antibody refers to any immunoglobulin that binds specifically to an antigenic determinant, and specifically binds to proteins identical or structurally related to the antigenic determinant that stimulated their production. Thus, antibodies can be useful in assays to detect the antigen that stimulated their production.
  • Monoclonal antibodies are derived from a single clone of B lymphocytes (i.e., B cells), and are generally homogeneous in structure and antigen specificity. Polyclonal antibodies originate from many different clones of antibody-producing cells, and thus are heterogenous in their structure and epitope specificity, but all recognize the same antigen.
  • antibody encompass any immunoglobulin (e.g., IgG, IgM, IgA, IgE, IgD, etc.) obtained from any source (e.g., humans, rodents, non-human primates, lagomorphs, caprines, bovines, equines, ovines, etc.).
  • immunoglobulin e.g., IgG, IgM, IgA, IgE, IgD, etc.
  • the term “antigen” is used in reference to any substance that is capable of being recognized by an antibody.
  • the terms “Western blot,” “Western immunoblot” “immunoblot” and “Western” refer to the immunological analysis of protein(s), polypeptides or peptides that have been immobilized onto a membrane support.
  • the proteins are first resolved by polyacrylamide gel electrophoresis (i.e., SDS-PAGE) to separate the proteins, followed by transfer of the protein from the gel to a solid support, such as nitrocellulose or a nylon membrane.
  • the immobilized proteins are then exposed to an antibody having reactivity towards an antigen of interest.
  • the binding of the antibody i.e., the primary antibody
  • the secondary antibody is typically conjugated to an enzyme that permits visualization of the antigen-antibody complex by the production of a colored reaction product or catalyzes a luminescent enzymatic reaction (e.g., the ECL reagent, Amersham).
  • compound refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function.
  • Compounds comprise both known and potential therapeutic compounds.
  • Compounds comprise polypeptides such as those described herein.
  • the term “toxic” refers to any detrimental or harmful effects on a subject, a cell, or a tissue as compared to the same cell or tissue prior to the administration of the toxicant.
  • composition refers to the combination of an active agent (e.g., Lunasin) with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • active agent e.g., Lunasin
  • compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.
  • topically refers to application of the compositions of the present invention to the surface of the skin and mucosal cells and tissues (e.g., alveolar, buccal, lingual, masticatory, or nasal mucosa, and other tissues and cells that line hollow organs or body cavities).
  • mucosal cells and tissues e.g., alveolar, buccal, lingual, masticatory, or nasal mucosa, and other tissues and cells that line hollow organs or body cavities.
  • the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintrigrants (e.g., potato starch or sodium starch glycolate), and the like.
  • the compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants. (See e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975), incorporated herein by reference).
  • gene refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA).
  • the polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length or fragment are retained.
  • the term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5′ and 3′ ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA. Sequences located 5′ of the coding region and present on the mRNA are referred to as 5′ non-translated sequences. Sequences located 3′ or downstream of the coding region and present on the mRNA are referred to as 3′ non-translated sequences.
  • the term “gene” encompasses both cDNA and genomic forms of a gene.
  • a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.”
  • Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript.
  • mRNA messenger RNA
  • RNA expression and “expression” refer to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via the enzymatic action of an RNA polymerase), and for protein encoding genes, into protein through “translation” of mRNA.
  • Gene expression can be regulated at many stages in the process.
  • Up-regulation” or “activation” refer to regulation that increases and/or enhances the production of gene expression products (e.g., RNA or protein), while “down-regulation” or “repression” refer to regulation that decrease production.
  • Molecules e.g., transcription factors
  • activators e.g., transcription factors
  • promoter/regulatory sequence means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence.
  • this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • cell culture refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro.
  • in vitro refers to an artificial environment and to processes or reactions that occur within an artificial environment.
  • in vitro environments can consist of, but are not limited to, test tubes and cell culture.
  • in vivo refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.
  • amino acid refers to any of the naturally occurring amino acids having the standard designations listed in Table 1, below. It also refers to those known synthetic amino acids. Unless otherwise indicated, all amino acid sequences listed in this disclosure are listed in the order from the amino terminus to the carboxyl terminus. As used herein, the abbreviations for any protective groups, amino acids and other compounds, are in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature, unless otherwise indicated (see Biochemistry 11: 1726 (1972)). As used herein, amino acid residues are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table:
  • peptide As used herein, the terms “peptide,” “polypeptide” and “protein” all refer to a primary sequence of amino acids that are joined by covalent “peptide linkages.” In general, a peptide consists of a few amino acids, typically from 2-50 amino acids.
  • polypeptide encompasses peptides and proteins, wherein the term “protein” typically refers to large polypeptides and the term “peptide” typically refers to short polypeptides.
  • the peptide, polypeptide or protein is synthetic, while in other embodiments, the peptide, polypeptide or protein is recombinant or naturally occurring.
  • a “synthetic” peptide is a peptide that is produced by artificial means in vitro (i.e., was not produced in vivo).
  • the term “peptide” further includes modified amino acids (whether naturally or non-naturally occurring), such modifications including, but not limited to, phosphorylation, glycosylation, pegylation, lipidization and methylation.
  • isolated peptide is a peptide which has been substantially separated from components (e.g., DNA, RNA, other proteins and peptides, carbohydrates and lipids) which naturally accompany it in a cell.
  • components e.g., DNA, RNA, other proteins and peptides, carbohydrates and lipids
  • the term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity or more (e.g., 99% sequence identity). Preferably, residue positions which are not identical differ by conservative amino acid substitutions.
  • the phrase “functionally equivalent” means that the variant, analogue or fragment of a polypeptide retains a desired biological activity in common with the lunasin polypeptide.
  • the desired biological activity in common with lunasin is biological activity related to the control, stabilization, or reduction in production or existing levels of cholesterol, LDL cholesterol, total cholesterol, or lipids.
  • a given quantity of the analogue, variant or fragment is at least 10%, preferably at least 30%, more preferably at least 50, 60, 80, 90, 95 or 99% as effective as an equivalent amount of the naturally occurring lunasin from which the analogue, variant or fragment is derived.
  • Determination of the relative efficacy of the analogue, variant or fragment can readily be carried out by utilizing a prescribed amount of the analogue, variant or fragment in the one or more of the assay methods of the invention and then comparing the ability of the analogue, variant or fragment to naturally occurring lunasin in tests that measure the ability of the sample to inhibit the acetylation of histone H3, or to effect the expression of HMG Co-A reductase, Sp1 or LDL-receptor.
  • analogue as used herein with reference to a polypeptide means a polypeptide which is a derivative of the polypeptide of the invention, which derivative comprises addition, deletion, and/or substitution of one or more amino acids, such that the polypeptide retains substantially the same function as the lunasin polypeptide identified below.
  • fragment refers to a polypeptide molecule that is a constituent of the full-length lunasin polypeptide and possesses qualitative biological activity in common with the full-length lunasin polypeptide.
  • the fragment may be derived from the full-length lunasin polypeptide or alternatively may be synthesized by some other means, for example chemical synthesis.
  • fragments it is intended to encompass fragments of a protein that are of at least 5, preferably at least 10, more preferably at least 20 and most preferably at least 30, 40 or 50 amino acids in length and which are functionally equivalent to the protein of which they are a fragment.
  • variant refers to a polypeptide which is produced from a nucleic acid encoding lunasin, but differs from the wild type lunasin in that it is processed differently such that it has an altered amino acid sequence.
  • a variant may be produced by an alternative splicing pattern of the primary RNA transcript to that which produces wild type lunasin.
  • Analogues and variants are intended to encompass proteins having amino acid sequence differing from the protein from which they are derived by virtue of the addition, deletion or substitution of one or more amino acids to result in an amino acid sequence that is preferably at least 60%, more preferably at least 80%, particularly preferably at least 85, 90, 95, 98, 99 or 99.9% identical to the amino acid sequence of the original protein.
  • the analogues or variants specifically include polymorphic variants and interspecies analogues.
  • the analogues and variants of the present invention further may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties.
  • One type of conservative amino acid substitution refers to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. More rarely, a variant may have “non-conservative” changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNAStar software. Variants can be tested in functional assays such as those described in the Examples section below.
  • conservative amino acid substitution refers to a substitution or replacement of one amino acid for another amino acid with similar properties within a polypeptide chain (primary sequence of a protein). For example, the substitution of the charged amino acid glutamic acid (Glu) for the similarly charged amino acid aspartic acid (Asp) would be a conservative amino acid substitution.
  • soybean lunasin refers to the natural, synthetically or recombinantly obtained soybean lunasin polypeptide set forth in (SEQ. ID. 2).
  • Guidance can be found for the identification and screening of functionally equivalent fragments and analogues of Lunasin peptide in the following references: U.S. Pat. No. 6,107,287, U.S. Pat. No. 6,544,956, US Patent Application 2003/0229038, filed Nov. 22, 2002, U.S. Pat. No. 6,391,848, U.S. patent application Ser. No. 10/252,256, filed Sep. 23, 2002, International Application WO 01/72784, filed Mar. 23, 2001, and U.S. patent application Ser. No. 10/302,633, filed Nov. 22, 2002, all of which are hereby incorporated by reference herein in their entirety for all purposes. These disclosures will guide one skilled in the art in identifying functionally equivalent and biologically active fragments, variants and analogues of lunasin.
  • “lunasin enriched” refers to compositions containing biologically active levels of naturally occurring lunasin, or a naturally occurring analogue of lunasin, that is at a concentration greater than that at which lunasin is found in the material used as the source of that lunasin or analogue.
  • “lunasin enriched seed extract” refers to compositions containing biologically active levels of naturally occurring lunasin, or a naturally occurring analogue of lunasin, that is at a concentration at least twice than that at which lunasin is naturally found in the source seed.
  • lunasin enriched compositions can be obtained from soybean, wheat, barley, soy isolates, soy concentrates, or other soy derived products, whether or not commercially obtained.
  • soy flour refers to soy flour compositions comprising soy flour and an amount of a protease inhibitor sufficient to protect lunasin, or a analogue, variant or fragment thereof, from complete digestion, wherein the compositions do not have levels of anti-nutritional elements that would cause an adverse effect in an individual who ingested them.
  • digestive material refers to the treatment of a polypeptide with a digestive material that breaks it down into its component amino acids.
  • digestive materials that can be used are well known in the art, and include, without limitation, pancreatin and other proteases such as trypsin, chymotrypsin, pepsin, Proteinase K, thermolysin, thrombin, Arg-C proteinase, Asp-N endopeptidase, AspN endopeptidase+N-terminal Glu, BNPS-Skatole, CNBr, clostripain, formic acid, glutamyl endopeptidase, iodosobenzoic acid, LysC, LysN, NTCB (2-nitro-5-thiocyanobenzoic acid), and Staphylococcal peptidase.
  • partially digested biologically active in relation to a polypeptide refers to the treatment of a polypeptide with a digestive material under conditions that increase the biological activity of the polypeptide.
  • composition therapy embraces the administration of a composition of the present invention in conjunction with another pharmaceutical agent that is indicated for treating or preventing a disorder, as part of a specific treatment regimen intended to provide a beneficial effect from the co-action of these therapeutic agents.
  • compositions herein may comprise, consist essentially of, or consist of any of the elements as described herein.
  • the present invention relates generally to a class of peptides that provide mammals with a variety of health related benefits. More specifically, the present invention involves using soy peptides to inhibit H3 acetylation, reduce expression of HMG-CoA reductase and increase LDL receptor and Sp1 expression in a mammal, protect against, prevent, or reduce: 1) the expression of Matrix metalloproteinase (MMP-1), 2) collagen breakdown, 3) photoaging and 4) the formation of skin wrinkles.
  • MMP-1 Matrix metalloproteinase
  • a method of inhibiting PCAF from acetylating H3 in a mammal includes providing an effective amount of lunasin peptides to a mammal to inhibit H3 acetylation in the mammal.
  • a method of reducing expression of HMG-CoA reductase in a mammal includes providing an effective amount of lunasin peptides to a mammal to reduce expression of HMG-CoA reductase in the mammal.
  • a method of increasing LDL receptor expression in a mammal includes providing an effective amount of lunasin peptides to a mammal to increase LDL receptor expression in the mammal.
  • a method of increasing Sp1 transcriptional activator expression in a mammal includes providing an effective amount of lunasin peptides to a mammal to increase Sp1 transcriptional activator expression in the mammal.
  • the effective amount of lunasin peptides that inhibit H3 acetylation, reduce expression of HMG-CoA reductase, increase LDL receptor expression or increases Sp1 transcriptional activator expression in a mammal is 25 to 100 mg daily.
  • the lunasin peptides include lunasin peptides or lunasin peptide derivatives.
  • the lunasin peptides are obtained from, soy, seed bearing plants other than soy, using recombinant DNA techniques and synthetic polypeptide production or any combination thereof.
  • the method includes providing an effective amount of one or more protease enzyme inhibitors to the lunasin peptides.
  • a method for protecting against photoaging of skin in an individual comprising: (a) providing: (i) an individual desiring to prevent photoaging of skin and, (ii) a composition comprising a compound selected from the group consisting of the peptide of SEQ ID NO 2 and a functionally equivalent variant, fragment or analogue of said peptide; and (b) administering said composition to said subject to protect against photoaging.
  • said compound is obtained from soybean, wheat or barley.
  • the compound is obtained by producing, extracting and purifying said compound using recombinant DNA techniques.
  • said compound is obtained by synthetic polypeptide production.
  • said individual is a human.
  • administering comprises topical administration of the composition.
  • the composition is in the form of a semi-solid formulation, liquid, gel, suspension, or aerosol spray.
  • said composition further comprises chymotrypsin inhibitor.
  • said compound is administered to said individual at between 5 ⁇ g/ml and 50 ⁇ g/ml.
  • a method for protecting against collagen breakdown in the skin in an individual comprising: (a) providing: (i) an individual desiring to prevent collagen breakdown in the skin and, (ii) a composition comprising a compound selected from the group consisting of the peptide of SEQ ID NO 2 and a functionally equivalent variant, fragment or analogue of said peptide; and (b) administering said composition to said subject to protect against collagen breakdown.
  • said compound is obtained from soybean, wheat or barley.
  • the compound is obtained by producing, extracting and purifying said compound using recombinant DNA techniques.
  • said compound is obtained by synthetic polypeptide production.
  • said individual is a human.
  • administering comprises topical administration of the composition.
  • the composition is in the form of a semi-solid formulation, liquid, gel, suspension, or aerosol spray.
  • said composition further comprises chymotrypsin inhibitor.
  • said compound is administered to said individual at between 5 ⁇ g/ml and 50 ⁇ g/ml.
  • a method for protecting against wrinkling of the skin in an individual comprising: (a) providing: (i) an individual desiring to prevent wrinkling of skin and, (ii) a composition comprising a compound selected from the group consisting of the peptide of SEQ ID NO 2 and a functionally equivalent variant, fragment or analogue of said peptide; and (b) administering said composition to said subject to protect against wrinkling of the skin.
  • said compound is obtained from soybean, wheat or barley.
  • the compound is obtained by producing, extracting and purifying said compound using recombinant DNA techniques.
  • said compound is obtained by synthetic polypeptide production.
  • said individual is a human.
  • administering comprises topical administration of the composition.
  • the composition is in the form of a semi-solid formulation, liquid, gel, suspension, or aerosol spray.
  • said composition further comprises chymotrypsin inhibitor.
  • said compound is administered to said individual at between 5 ⁇ g/ml and 50 ⁇ g/ml.
  • FIG. 1 shows the 2S albumin protein encoded by Gm2S 1 cDNA (SEQ ID NO 1). Arrows indicate endoproteolytic sites that give rise to small subunit (“lunasin”) (SEQ ID NO 2) and the large subunit (methionine rich protein). Important regions in both subunits are indicated.
  • FIG. 2 is a photograph of a Western blot analysis (top) and a table (below) showing densitometer values indicating the relative levels of expression of HMG-CoA reductase in HepG2 cells that were (CFM+LS (24)) or were not (CFM) treated with lunasin for 24 hours prior to incubation in cholesterol free media (CFM) for 24 hours to activate sterol regulatory element binding proteins (SREBP.) After incubations, total protein was extracted and 10 ug protein was loaded onto 10% Tris-glycine gels, electroblotted onto nitrocellulose membrane, and immunostained with primary antibodies raised against HMG-CoA reductase and actin (to show equal loading of proteins.) Spot densitometer values represent mean and standard deviation of data from three separate experiments.
  • FIG. 3 is a photograph of a Western blot analysis (top) and a table (below) showing densitometer values indicating the relative levels of expression of LDL receptor in HepG2 cells that were (CFM+LS(24)) or were not (CFM) treated with lunasin for 24 hours prior to incubation in cholesterol free media (CFM) for 24 hours to activate SREBP. After incubations, total protein was extracted and 10 ug protein was loaded onto 10% Tris-glycine gels, electroblotted onto nitrocellulose membrane, and immunostained with primary antibodies raised against LDL-receptor and actin (to show equal loading of proteins.) Spot densitometer values represent mean and standard deviation of data from three separate experiments.
  • FIG. 4 is a photograph of a Western blot analysis (top) and a table (below) showing densitometer values indicating the relative levels of expression of Sp1 in HepG2 cells that were grown from confluence in growth media for 24 hours before growth media was replaced with fresh growth media (Media), media with lunasin (Media+LS) or cholesterol free media with lunasin (CFM+LS) or without lunasin (CFM). Samples were then incubated for 24 or 48 hours as indicated.
  • Media fresh growth media
  • Media+LS media with lunasin
  • CFM+LS cholesterol free media with lunasin
  • CFM lunasin
  • FIG. 5 shows the western blots from experiments on PCAF reaction products demonstrating that lunasin caused a dramatic reduction in histone H3 acetylation.
  • Acid extracted protein from untreated (untrt) HeLa cells was used as template in histone acetylase reactions using HAT enzyme, PCAF, in the presence or absence of 10 uM lunasin.
  • Reaction products were immunoblotted and stained with antibodies against diacetylated histone H3.
  • Untrt ( ⁇ ) is the histone template control, NaB (+) correspond to acid extracted histones from NaButyrate treated HeLa cells (positive control). Boxed signal indicates significant decrease in H3 acetylation upon addition of 10 uM lunasin compared with no lunasin application. Numbers in parenthesis indicate densitometer readings relative to the untreated control (set as 1) in PCAF HAT reaction products.
  • FIG. 6 shows the western blots from experiments on PCAF HAT reaction products demonstrating that lunasin caused a dramatic reduction in histone H3 acetylation.
  • Acid extracted histones isolated from untreated (untrt) HeLa cells were used in PCAF HAT reactions, immunoblotted and stained with antibodies to H3 Ac-Lys9 and H3 Ac-Lys14.
  • Untrt ( ⁇ ) is the histone template control
  • NaB (+) correspond to acid extracted histones from NaButyrate treated HeLa cells (positive control)
  • +Lun correspond to 10 uM lunasin treated histone template
  • ⁇ Lun correspond to non-lunasin treated.
  • Boxed signal indicate decreased H3 Lys 14 acetylation by PCAF acetylase enzyme in the presence of lunasin. Numbers in parenthesis indicate densitometer readings relative to the signal level of lunasin/lunasin treatment (set as 1) in immunoblots stained with Ac-Lys14H3.
  • Lunasin is the first natural substance to be identified as a histone acetylase inhibitor, although it does not directly affect the histone acetylase enzyme. It inhibits H3 and H4 acetylation by binding to specific deacetylated lysine residues in the N-terminal tail of histones H3 and H4, making them unavailable as substrates for histone acetylation.
  • the elucidation of the mechanism of action makes lunasin an important molecule for research studies to understand the emerging role of epigenetics and chromatin modifications in important biological processes.
  • This biological model was chosen because activation of SREBPs by sterol depletion results in the increased acetylation of histone H3 but not histone H4, by the histone acetylase enzyme PCAF, in chromatin proximal to the promoters of HMG-CoA reductase and the LDL receptor genes (8) and SREBP activation results in the increased recruitment of co-regulatory factors, CREB to the promoter of HMG-CoA reductase gene, and Sp1 to the promoter of LDL receptor gene (8).
  • lunasin (a.k.a. lunastantin) is the bioactive agent from soy responsible for inhibiting H3 acetylation, reducing expression of HMG-CoA reductase and increasing LDL receptor and Sp1 expression in a mammal.
  • FIG. 1 shows the 2S albumin protein and the small lunasin subunit. It has been shown that constitutive expression of the lunasin gene in mammalian cells disturbs kinetochore formation and disrupts mitosis, leading to cell death (2). When applied exogenously in mammalian cell culture, the lunasin peptide suppresses transformation of normal cells to cancerous foci that are induced by chemical carcinogens and oncogenes.
  • lunasin (a) is internalized through its RGD cell adhesion motif, (b) colocalizes with hypoacetylated chromatin in telomeres at prometaphase, (c) binds preferentially to deacetylated histone H4, which is facilitated by the presence of a structurally conserved helical motif found in other chromatin-binding proteins, (d) inhibits histone H3 and H4 acetylation, and (e) induces apoptosis in E1A-transfected cells (4).
  • a method of inhibiting H3 acetylation in a mammal includes providing an effective amount of lunasin peptides to a mammal to inhibit H3 acetylation in the mammal.
  • a method of reducing expression of HMG-CoA reductase in a mammal includes providing an effective amount of lunasin peptides to a mammal to reduce expression of HMG-CoA reductase in the mammal.
  • a method of increasing LDL receptor expression in a mammal includes providing an effective amount of lunasin peptides to a mammal to increase LDL receptor expression in the mammal.
  • a method of increasing Sp1 transcriptional activator expression in a mammal includes providing an effective amount of lunasin peptides to a mammal to increase Sp1 transcriptional activator expression in the mammal.
  • the effective amount of lunasin peptides that inhibit H3 acetylation, reduce expression of HMG-CoA reductase, increase LDL receptor expression or increases Sp1 transcriptional activator expression in a mammal is 25 to 100 mgs daily. It should be appreciated that the effective amount of lunasin will depend, at least in part, on the size, weight, health and desired goals of the mammals consuming the compositions. Accordingly, it is believed that in at least one embodiment, the effective amount of lunasin provided to the mammal is 25 mg to 100 mg daily.
  • the lunasin peptides include lunasin peptides or lunasin peptide derivatives. It should also be appreciated that the present invention includes the use of lunasin peptide derivatives, which are any peptides that contain the same functional units as lunasin. It should also be appreciated the products and compositions of the present invention can be used in, foods, powders, bars, capsules, shakes and other well known products consumed by mammals or used separately.
  • the lunasin peptides are obtained from, soy, seed bearing plants other than soy, using recombinant DNA techniques and synthetic polypeptide production or any combination thereof.
  • the method includes providing an effective amount of one or more protease enzyme inhibitors with or without the lunasin peptides.
  • the protease enzyme inhibitors act to protect lunasin from digestion and facilitate absorption and delivery to the appropriate target areas.
  • appropriate protease enzyme inhibitors include, but are not limited to, pancreatin, trypsin and/or chymotrypsin inhibitors. It should be appreciated that the scope of the present inventions includes the use of the lunasin and/or lunasin derivatives with any other composition or product that is known or believed to facilitate lunasin's absorption or delivery in a mammal.
  • the activity of lunasin peptides in prevention of histone H3 acetylation has additional applications for the protection against and prevention of the expression of Matrix metalloproteinase (MMP-1), collagen breakdown, photoaging and the formation of skin wrinkles.
  • MMP-1 Matrix metalloproteinase
  • the present invention encompasses the use of lunasin peptides for the protection against and prevention of the expression of Matrix metalloproteinase (MMP-1), collagen breakdown in the skin, photoaging of the skin, premature aging of the skin and the formation of skin wrinkles.
  • MMP-1 Matrix metalloproteinase
  • HAT histone acetylase
  • MMP-1 Matrix metalloproteinase
  • HAT inhibitors reduces UV-induced expression of MMP-1 (20). It is thus desirable to interfere with the acetylation of histone H3 in order to reduce the expression of MMP-1 and as a result reduce collagen breakdown and the photoaging and the premature aging associated with it.
  • compositions can be administered using a number of different routes including oral administration, topical administration, transdermal administration, or injection directly into the body.
  • Administration of compositions for use in the practice of the present invention can be systemic (i.e., administered to the subject as a whole via any of the above routes) or localized (i.e., administered to the specific location of the particular disease or pathological condition of the subject via any of the above routes).
  • the compositions to decrease the expression of Matrix metalloproteinase (MMP-1), protect against collagen breakdown in the skin, protect against skin photoaging, or protect against skin wrinkling in an individual are administered by topical administration.
  • MMP-1 Matrix metalloproteinase
  • kits, and compositions can also be used in “combination therapy” with another composition or treatment that is indicated for treating or preventing a disorder.
  • a product containing an effective amount of lunasin peptides to prevent the expression of Matrix metalloproteinase (MMP-1), reduce collagen breakdown, or prevent dermal photoaging in an individual that is treated with the product is provided.
  • MMP-1 Matrix metalloproteinase
  • compositions of the present invention can be administered in various doses to provide effective treatment concentrations based upon the teachings of the present invention.
  • Factors such as the activity of the selected compositions, the physiological characteristics of the subject, the extent or nature of the subject's disease or pathological condition, and the method of administration will determine what constitutes an effective amount of the selected compositions.
  • initial doses will be modified to determine the optimum dosage for treatment of the particular subject.
  • Suitable dosages can be chosen by taking into account any or all of such factors as the size, weight, health, age, and sex of the human or individual, the desired goals of the patient, the severity of the pathological condition for which the composition is being administered, the response to treatment, the type and quantity of other medications being given to the patient that might interact with the composition, either potentiating it or inhibiting it, and other pharmacokinetic considerations such as liver and kidney function. These considerations are well known in the art and are described in standard textbooks.
  • a therapeutically effective amount of any embodiment of the present invention is determined using methods known to pharmacologists and clinicians having ordinary skill in the art. Blood levels of the composition can be determined using routine biological and chemical assays and these blood levels can be matched to the route of administration. The blood level and route of administration giving the most desirable level of cholesterol reduction can then be used to establish an “effective amount” of the pharmaceutical composition for treatment.
  • compositions of the present invention can be used to ascertain a therapeutically effective amount of the compositions of the present invention for treating any and all disorders described herein.
  • animal models as described below can be used to determine applicable dosages to treat or prevent a particular disease or pathological condition.
  • dosage-effect relationships from in vitro or in vivo tests initially can provide useful guidance on the proper doses for subject administration.
  • methods and compositions of the invention encompass a dose of a composition comprising lunasin, or a functionally equivalent variant, analogue or fragment of lunasin, of about 5 ng to about 1000 g, or about 100 ng to about 600 mg, or about 1 ⁇ g to about 500 ⁇ g, or about 5 ⁇ g/ml and 50 ⁇ g/ml.
  • a dosage unit of a composition of the present invention can typically contain, for example, without limitation, about 5 ng, 50 ng 100 ng, 500 ng, 1 ⁇ g, 10 ⁇ g, 100 ⁇ g, 250 ⁇ g, 500 ⁇ g, 1 mg, 10 mg, 20 mg, 40 mg, 80 mg, 100 mg, 125 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 20 g, 30 g, or 40 g of a composition of the present invention.
  • compositions of the present invention contain about 1 to 500 ⁇ g, preferably 5 ⁇ g/ml and 50 ⁇ g/ml, more preferably approximately 250 ⁇ g per dosage of lunasin, or fragments, variants and analogues of lunasin.
  • Exemplary dosages for lunasin, or fragments, variants and analogues thereof, in accordance with the teachings of the present invention range from 0.1 ⁇ g to 200 mg, preferably, 1 ⁇ g to 100 mg, more preferably 25 ⁇ g to 500 ⁇ g for humans and other individuals, although alternative dosages are contemplated as being within the scope of the present invention.
  • lunasin, or fragments, variants and analogues thereof is present at a level of between 25 ⁇ g/ml and 25 mg/ml, more preferably between 50 ⁇ g/ml and 1 mg/ml, more preferably between 100 ⁇ g/ml and 500 ⁇ g/ml, even more preferably, approximately 250 ⁇ g/ml.
  • MMP-1 Matrix metalloproteinase
  • compositions and methods for oral administration is provided to an individual at a level of between 0.01 mg/Kg and 100 mg/Kg body weight of an individual, preferably 0.05 mg/Kg and 50 mg/Kg, more preferably between 0.5 mg/Kg and 2.5 mg/Kg, and even more preferably between 0.2 mg/Kg and 1.5 mg/Kg.
  • a dose can be administered in one to about four doses per day, or in as many doses per day to elicit a therapeutic effect.
  • the dosage form can be selected to accommodate the desired frequency of administration used to achieve the specified dosage, as well as the route of delivery.
  • the amount of therapeutic agent necessary to elicit a therapeutic effect can be experimentally determined based on, for example, the absorption rate of the agent into the blood serum, or the dermal layer of the skin for topical applications, and the bioavailability of the agent. Determination of these parameters is well within the skill of the art.
  • the invention also concerns formulations containing the compositions of the present invention.
  • the products and compositions of the present invention can be used alone or in foods, powders, bars, capsules, shakes and other well known products consumed by individuals.
  • compositions of the present invention are together with a dietary suitable excipient, diluent, carrier, or with a food.
  • the formulation is in the form of a pill, tablet, capsule, powder, food bar or similar dosage form.
  • the formulations may be a variety of kinds, such as nutritional supplements, pharmaceutical preparations, vitamin supplements, food additives or foods supplemented with the specified compositions of the invention, liquid or solid preparations, including drinks, sterile injectable solutions, tablets, coated tablets, capsules, powders, drops, suspensions, or syrups, ointments, lotions, creams pastes, gels, or the like.
  • compositions of the present invention may be packaged in convenient dosage forms, and may also include other active ingredients, and/or may contain conventional excipients, pharmaceutically acceptable carriers and diluents.
  • compositions of the present invention in herbal remedies and treatments is also a preferred part of the invention.
  • Topical formulations typically are gels, salves, powders, or liquids, though controlled formulations which release defined amounts of active ingredient at the desired surface are also desirable.
  • the formulations may contain materials which enhance the permeability of the active moieties through the epidermis.
  • penetrants include, for example, DMSO, various bile salts, non-toxic surfactants and the like.
  • Standard ingredients for cosmetic/pharmaceutical compositions are well known in the art; formulations for topical application of pharmaceuticals are found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, Pa., incorporated herein by reference. Cosmetic formulations are widely varied and well known to practitioners.
  • statin drugs The lowering of serum cholesterol by statin drugs is achieved by competitively inhibiting the HMG-CoA reductase, the rate limiting enzyme in the body's metabolic pathway for synthesis of cholesterol.
  • statins By reducing endogenous cholesterol synthesis, statins also cause liver cells to up regulate expression of the LDL receptor, leading to increased clearance of low-density lipoprotein (LDL) from the bloodstream (9).
  • LDL low-density lipoprotein
  • SREBP Sterol Regulatory Element-Binding Protein-1 and -2
  • HepG2 cells (1 ⁇ 10 6 ) were treated with or without 10 uM synthetic lunasin in DMEM with 10% FBS for 24 hours before growth media was replaced with cholesterol-free media to activate SREBP. After 24 hours, total protein was extracted and 10 ug protein was loaded onto 10% Tris-glycine gels, electroblotted onto nitrocellulose membrane, and immunostained with primary antibodies raised against HMG-CoA reductase and actin (to show equal loading of proteins). Spot densitometer values are obtained by digital scanning and Un-Scan It software, and represent mean and standard deviation of data from three separate experiments. The results are shown in FIG. 2 .
  • HepG2 cells (1 ⁇ 10 6 ) were treated with or without 10 uM synthetic lunasin in DMEM with 10% FBS for 24 hours before growth media is replaced with cholesterol-free media to activate SREBP. After 24 hours, total protein was extracted and 10 ug proteins loaded onto 10% Tris-glycine gels, electroblotted onto nitrocellulose membrane, and immunostained with primary antibodies raised against LDL-receptor and actin (to show equal loading of proteins). Spot densitometer values were obtained by digital scanning and Un-Scan It software, and represent mean and standard deviation of data from three separate experiments. The results are shown in FIG. 3 .
  • FIGS. 2 and 3 show the upregulation of HMG-CoA reductase (98% increase) and LDL-receptor (34% increase) when HepG2 cells are grown in cholesterol-free media for 24 hours.
  • lunasin is added to the cholesterol-free media, the expression of the HMG-CoA reductase is reduced by more than 50% ( FIG. 2 ), while the expression of LDL-receptor has increased by more than 60% ( FIG. 3 ).
  • This effect of lunasin is similar to statin drugs that reduces endogenous cholesterol synthesis by inhibiting HMG-CoA reductase activity, which leads to increased LDL receptor expression.
  • statin drugs that reduces endogenous cholesterol synthesis by inhibiting HMG-CoA reductase activity, which leads to increased LDL receptor expression.
  • the mode of action of lunasin is believed to differ from statin drugs in that it appears to inhibit expression of HMG-CoA reductase at the transcriptional level, rather than on inhibiting its enzyme activity.
  • lunasin up regulates the expression of LDL-receptor gene.
  • SREBP activation of LDL-receptor by sterol depletion requires increased recruitment of Sp1 co-activator to a site adjacent to SREBP in the promoter/regulatory sequence of LDL-receptor gene (25).
  • the up regulation of LDL-receptor by lunasin (LS) in cholesterol-free media may be due to increased availability and recruitment of the Sp1 coactivator to the LDL-receptor promoter/regulatory sequence.
  • the level of Sp1 was determined in lunasin-treated growth media and cholesterol-free media by Western analysis using Sp1 antibody, as follows: HepG2 cells (1 ⁇ 10 6 ) were grown from confluence in DMEM with 10% FBS for 24 hours before growth media was replaced with fresh growth media or cholesterol-free media (to activate SREBP) and treated with, or without 10 uM synthetic lunasin. After 24 hours, total protein was extracted from each treatment and 10 ug protein loaded onto 10% Tris-glycine gels, electroblotted onto nitrocellulose membrane, and immunostained with primary antibodies raised against Sp1 and actin (to show equal loading of proteins). Spot densitometer values were obtained by digital scanning and Un-Scan It software and represent data from one experiment. The results are shown in FIG. 4 .
  • FIG. 4 shows that Sp1 levels in control and lunasin-treated growth media were not significantly different. However, Sp1 levels increased in cholesterol-free media by 23%, compared to the growth media. The addition of lunasin in the cholesterol-free media further increased Sp1 levels by almost 60%, which closely mirrors the increase in LDL-receptor levels in lunasin-treated, cholesterol-free media.
  • the data shows that lunasin inhibits the expression of HMG-CoA reductase, the rate limiting enzyme in the body's metabolic pathway for synthesis of cholesterol, and at the same time increases the expression of the LDL receptor, leading to increased clearance of low-density lipoprotein (LDL) from the bloodstream, which will lower total and LDL cholesterol in a mammal.
  • LDL low-density lipoprotein
  • the other (II) is electroblotted onto nitrocellulose membrane and incubated with affinity-purified lunasin polyclonal antibody (Pacific Immunology, Ramona, Calif.) followed by HRP-conjugated donkey anti-rabbit secondary antibody (Amersham Biosciences, Piscataway, N.J.).
  • lunasin polyclonal antibody Pacific Immunology, Ramona, Calif.
  • HRP-conjugated donkey anti-rabbit secondary antibody Amersham Biosciences, Piscataway, N.J.
  • Lunasin immunosignals are detected using the ECL Western blotting kit from Amersham.
  • Formulated lunasin-enriched soy concentrate (LeSC) and LeSC supplemented with soy flour (SF) contain significant amounts of lunasin. This experiment evaluated the amount of lunasin in lunasin-enriched soy concentrate (LeSC) and LeSC supplemented with soy flour.
  • a composition comprising lunasin enriched soy concentrate and soy flour.
  • compositions of the present invention that comprise naturally derived lunasin can be optimized for use in particular methods of the present invention by varying the amount of total protein and lunasin content, which can be controlled by the amount of soy concentrate used, and varying the amount of lunasin protection from digestion, which can be controlled by the amount of minimally heated soy flour used.
  • a composition comprising lunasin and soy flour is optimized through preparation methods describe herein or known to one skilled in the art, to have a level of protease inhibitors sufficient to protect lunasin biological activity during digestion but not sufficient to have levels of anti-nutritional elements that are undesirable for oral use.
  • the present invention teaches improved methods of determining lunasin concentration in starting materials and final products of the present invention, so as to maximize the concentration of lunasin and therefore the activity of compositions of treatment in cholesterol related applications.
  • the ratio of soy flour to soy concentrate is between 10:90 and 50:50, more preferably between 20:80 and 40:60, more preferably approximately 30:70 soy flour:soy concentrate. This ratio for minimally heated soy flour and soy concentrate was determined to provide a biologically active concentration of lunasin and as well as sufficient protection from digestion by the soy flour.
  • the Western blotting analysis procedure used in this experiment was as follows: approximately 20 ug of total protein from LeSC, SF and the LeSC+SF were electrophoresed in 16% Tris-Tricine gels and electroblotted onto nitrocellulose membrane. Blots were incubated with lunasin polyclonal antibody followed by HRP-conjugated anti-rabbit secondary antibody before lunasin immunosignals were detected with the ECL kit. The results showed that both LeSC and LeSC+SF contained significant amounts of lunasin.
  • Synthetic lunasin reduced acetylation of histone H3 by the histone acetylase enzyme, PCAF, using core histones isolated from chicken erythrocyte (Upstate/Millipore, Billerica, Mass.) as template for the HAT assay.
  • PCAF histone acetylase enzyme
  • Partial digestion of formulated LeSC+SF increases biological activity of lunasin.
  • a confirmatory experiment to determine the biological activity of digested and undigested LeSC and LeSC+SF was conducted using a different core histone template. This time we used the core histones extracted from HeLa tumor cells. Unlike the chicken erythrocyte cells, core histones from sodium butyrate treated HeLa cells are commercially available (Upstate/Millipore, Piscataway, N.J.), and can be used as a positive control for histone acetylation. The core histones isolated from untreated HeLa cells were used as a negative control (low levels of histone acetylation) and as template for the HAT assay.
  • the HAT bioactivity assay was conducted using acid extracted core histones from HeLa cells (Upstate/Millipore) as a template (temp ( ⁇ ) control) for the PCAF catalyzed HAT reaction.
  • Core histones from sodium butyrate (NaB) treated HeLa cells were used as a positive control since NaB is a histone deacetylase inhibitor known to increase histone acetylation.
  • the inhibitory effect of synthetic lunasin (+synL) on histone H3 acetylation by PCAF was used to compare the effect of lunasin-enriched soy concentrate (A), digested LeSC (A dig), LeSC+SF (B) and digested LeSC+SF (B dig).
  • LeSC and LeSC+SF were partially digested by adding pancreatin at 1:0.5 (w/w) and incubating at 38° C. for 15 min.
  • the numbers below the legend indicate relative densitometer readings normalized using immunosignal from the template (temp). Low numbers indicate presence of lunasin biological activity.
  • LeSC+SF was partially digested by mixing it with freshly prepared pancreatin solution (10 ⁇ g/mL of distilled water) in a 1:0.5, (w/w) ratio. Mixture was incubated at 38° C. for 15 min. before proteases and digestive enzymes were inactivated by boiling for 5 min and then quenching in ice. Under these digestion conditions the lunasin in the LeSC soy extract was digested and inactivated while that of LeSC+SF were more biologically active.
  • the conditions for the partial digestion of LeSC+SF has to be determined empirically by analyzing digestion products for lunasin content and biological activity using the HAT assay.
  • Variations in the sources of pancreatin and protease enzymes, the age of the protease enzyme, or incubation conditions can lead to variability in digestion conditions. For example, the use of one month old preparations of pancreatin for partial digestion led to the degradation and loss of activity of lunasin under similar incubation conditions described above. Therefore, in a preferred embodiment of the present invention, acceptable ranges for concentration of and incubation time with the protease enzymes are determined using an assay such as the HAT assay used above to evaluate biological activity of the treated compositions.
  • Chymotrypsin inhibitors protect the bioactivity of lunasin. To determine which protease inhibitors found in soy protects lunasin from digestion, soybean trypsin inhibitor and trypsin+chymotrypsin inhibitors were obtained from Sigma and mixed with LeSC on 1:1 w/w ratio. The mixtures were digested with pancreatin, and digestion products immunostained with lunasin antibody.
  • HAT bioactivity assay was conducted using core histones from chicken erythrocyte cells (Upstate/Millipore) as a template for the PCAF catalyzed HAT reaction.
  • the inhibitory effect of synthetic lunasin (+synL) on histone H3 acetylation by PCAF as compared to the negative untreated control ( ⁇ synL) was used to compare the effect of digested LeSC (A), digested LeSC+try+chy (B), digested LeSC+try (C), undigested LeSC (D) and undigested LeSC+SF (E.)
  • Core histones purified from chicken erythrocyte cells were used as templates in histone acetylase (HAT) reactions using PCAF histone acetylase enzyme, in the presence or absence of around 2-10 uM lunasin.
  • the core histone template and lunasin-enriched soy concentrates (LeSC and LeSC+SF) were mixed (10:1 w/w) and incubated in ice for 5 min and 25° C. for 10 min before mixture was added to 1 ⁇ HAT reaction mix, 1 uM acetyl CoA and 5 uL PCAF (based on recommended concentration from Upstate/Millipore). Reaction mixture was incubated at 30° C. while shaking at 250 rpm for 1 h.
  • Reaction was stopped by adding Laemmli stop buffer (1:1 v/v) with beta-mercaptoethanol, and boiling for 5 min. before quenching in ice for 15 min.
  • the products of PCAF HAT reaction were run on 16% SDS-PAGE, blotted onto nitrocellulose membrane and immunostained with primary antibodies raised against diacetylated histone H3 (Ac-Lys 13+Ac-Lys14H3) followed by HRP-conjugated anti-rabbit secondary antibody.
  • Chemiluminescent signals from antibody complexes were visualized using standard chemiluminescent reagents and exposed to Kodak BioMAX film, developed and spot densitometer measured by using digital scanner and UN-SCAN-IT software program from Silk Scientific (Orem, Utah).
  • compositions as well as treatment utilization of kits and treatment methods, may be optionally determined by either of the following procedures.
  • Male dogs (beagles, ranging from about 9 to about 14 kilograms, 1 to 4 years old) are fed a standard dog feed supplemented with 5.5% lard and 1% cholesterol.
  • Baseline blood samples are drawn from fasted dogs prior to initiating the study to obtain reference values for plasma cholesterol. Dogs are then randomized to groups of five animals with similar plasma cholesterol levels. The animals are dosed in accordance with a treatment method described herein immediately prior to diet presentation for seven days. Blood samples are obtained 24 hours after the last dose for plasma cholesterol determinations. Plasma cholesterol levels are determined by a modification of the cholesterol oxidase method using a commercially available kit.
  • hamsters are separated into groups of six and given a controlled cholesterol diet containing 0.5% cholesterol for seven days. Diet consumption is monitored to determine dietary cholesterol exposure.
  • the animals are dosed in accordance with a treatment method described herein once daily beginning with the initiation of diet. Dosing is by oral gavage. All animals moribund or in poor physical condition are euthanized. After seven days, the animals are anesthetized by intramuscular (IM) injection of ketamine and sacrificed by decapitation. Blood is collected into vacutainer tubes containing EDTA for plasma lipid analysis and the liver is excised for tissue lipid analysis. Lipid analysis is conducted as per published procedures (e.g., Schnitzer-Polokoff et al., Comp. Biochem. Physiol., 99A, 4 (1991), pp. 665-670 and data is recorded as percent reduction of lipid versus control.
  • IM intramuscular

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WO2014145086A2 (en) * 2013-03-15 2014-09-18 Galvez Alfredo Flores Products and methods using lunasin-enriched soy extract mixtures to reduce free fatty acid levels, increase leptin levels and increase adiponectin levels in plasma
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US20110301091A1 (en) * 2008-11-03 2011-12-08 Giammaria Giuliani Compositions and methods for reducing the signs of aging of the skin
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CN104840943A (zh) * 2015-05-05 2015-08-19 中国农业科学院作物科学研究所 Lunasin多肽在制备具有减肥活性物质方面的应用
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CN107712914A (zh) * 2017-11-20 2018-02-23 中食都庆（山东）生物技术有限公司 一种具有降脂功能的复方产品及其制备方法
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US20100087367A1 (en) * 2006-09-15 2010-04-08 Soy Labs, Llc Products and methods using soy peptides to lower total and ldl cholesterol levels
US8598111B2 (en) * 2006-09-15 2013-12-03 Soy Labs, Llc Products and methods using soy peptides to lower total and LDL cholesterol levels
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