WO2013148282A1 - Methods and materials related to nutritional supplement compositions containing a potato polysaccharide preparation - Google Patents

Methods and materials related to nutritional supplement compositions containing a potato polysaccharide preparation Download PDF

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Publication number
WO2013148282A1
WO2013148282A1 PCT/US2013/031700 US2013031700W WO2013148282A1 WO 2013148282 A1 WO2013148282 A1 WO 2013148282A1 US 2013031700 W US2013031700 W US 2013031700W WO 2013148282 A1 WO2013148282 A1 WO 2013148282A1
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WO
WIPO (PCT)
Prior art keywords
potato polysaccharide
composition
polypeptide
polysaccharide preparation
potato
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Ceased
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PCT/US2013/031700
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French (fr)
Inventor
George B. Stefano
Richard Kream
Kirk J. MANITONE
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Research Foundation of the State University of New York
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Research Foundation of the State University of New York
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Application filed by Research Foundation of the State University of New York filed Critical Research Foundation of the State University of New York
Priority to HK15107744.7A priority Critical patent/HK1207082A1/en
Priority to US14/388,442 priority patent/US20150065451A1/en
Priority to AU2013240229A priority patent/AU2013240229B2/en
Priority to EP13769274.5A priority patent/EP2834255B1/en
Priority to CA2868921A priority patent/CA2868921C/en
Priority to JP2015503314A priority patent/JP6421112B2/en
Publication of WO2013148282A1 publication Critical patent/WO2013148282A1/en
Anticipated expiration legal-status Critical
Priority to US15/472,559 priority patent/US20170232033A1/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A23L19/10—Products from fruits or vegetables; Preparation or treatment thereof of tuberous or like starch containing root crops
    • A23L19/12—Products from fruits or vegetables; Preparation or treatment thereof of tuberous or like starch containing root crops of potatoes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/385—Heterocyclic compounds having sulfur as a ring hetero atom having two or more sulfur atoms in the same ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • 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)
    • A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
    • 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
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • 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
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the document relates to nutritional supplement compositions.
  • this document relates to nutritional supplement compositions containing a potato polysaccharide preparation, methods for obtaining potato polysaccharide preparations, methods for making nutritional supplement compositions containing a potato polysaccharide preparation, and methods for increasing or decreasing expression of polypeptides involved with mitochondria activity or function.
  • Potatoes are starchy, edible tubers obtained from potato plants and form an integral part of much of the world's food supply. In fact, potatoes are the fourth largest food crop in the world. The main potato species worldwide is Solanum tuberosum.
  • the document provides nutritional supplement compositions.
  • this document provides nutritional supplement compositions containing a potato polysaccharide preparation, methods for obtaining potato polysaccharide preparations, methods for making nutritional supplement compositions containing a potato polysaccharide preparation, and methods for increasing or decreasing expression of polypeptides involved with mitochondria activity or function.
  • the nutritional supplement compositions and potato polysaccharide preparations provided herein can be used to increase or decrease expression of polypeptides involved with mitochondria activity or function.
  • a nutritional supplement composition containing a potato polysaccharide preparation provided herein or a potato polysaccharide preparation provided herein can be used to increase expression of a transcription factor A, mitochondrial polypeptide (a TFAM polypeptide), an ATP synthase, H + transporting, mitochondrial Fl complex, alpha subunit 1 polypeptide (an ATP5A1 polypeptide), a pyruvate dehydrogenase (lipoamide) alpha 1 polypeptide (a PDHA1 polypeptide), a pyruvate dehydrogenase (lipoamide) alpha 2 polypeptide (a PDHA2 polypeptide), a thimet oligopeptidase 1 polypeptide (a THOP1 polypeptide), or a combination thereof.
  • a transcription factor A mitochondrial polypeptide
  • an ATP synthase H + transporting
  • mitochondrial Fl complex alpha subunit 1 polypeptide
  • an ATP5A1 polypeptide alpha subunit 1 polypeptide
  • a nutritional supplement composition containing a potato polysaccharide preparation provided herein or a potato polysaccharide preparation provided herein can be used to decrease expression of a forkhead box 01 polypeptide (a FOXOIA polypeptide), a nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 polypeptide (a NFKBl polypeptide), a pyruvate dehydrogenase kinase, isozyme 2 polypeptide (a PDK2 polypeptide), a pyruvate dehydrogenase kinase, isozyme 4 polypeptide (a PDK4 polypeptide), a 3- hydroxy-3-methylglutaryl-CoA reductase polypeptide (a HMGCR polypeptide), or a combination thereof.
  • a forkhead box 01 polypeptide a FOXOIA polypeptide
  • a nutritional supplement composition containing a potato polysaccharide preparation provided herein or a potato polysaccharide preparation provided herein can be used to increase one or more polypeptides (e.g., one or more of a TFAM polypeptide, an ATP5A1 polypeptide, a PDHA1 polypeptide, a PDHA2 polypeptide, or a THOP1 polypeptide) and decrease one or more polypeptides (e.g., one or more of a FOXOIA polypeptide, a NFKBl polypeptide, a PDK2 polypeptide, a PDK4 polypeptide, or a HMGCR polypeptide).
  • polypeptides e.g., one or more of a TFAM polypeptide, an ATP5A1 polypeptide, a PDHA1 polypeptide, a PDHA2 polypeptide, or a THOP1 polypeptide
  • polypeptides e.g., one or more
  • polysaccharide preparations provided herein can be used to increase or decrease expression of polypeptides involved with obesity and/or diabetes.
  • a nutritional supplement composition containing a potato polysaccharide preparation provided herein or a potato polysaccharide preparation provided herein can be used to increase expression of a lipase, hormone-sensitive polypeptide (an LIPE polypeptide) in adipocytes, to increase expression of a phosphoenolpyruvate carboxykinase 2 (mitochondrial) polypeptide (a PCK2 polypeptide), to increase expression of a monoacylglycerol O-acyltransferase 1 polypeptide (an MOGAT1 polypeptide), to increase expression of a peroxisome proliferator-activated receptor gamma, coactivator 1 alpha polypeptide (a PPARGCla polypeptide), to increase expression of a peroxisome proliferator-activated receptor gamma, coactivator 1 beta polypeptide (a PPAR
  • a nutritional supplement composition containing a potato polysaccharide preparation provided herein or a potato polysaccharide preparation provided herein can be used to decrease expression of a l-acylglycerol-3- phosphate O-acyltransferase 1 polypeptide (an AGP ATI polypeptide), to decrease expression of an oxidized low density lipoprotein (lectin-like) receptor 1 polypeptide (an OLR1 polypeptide), to decrease expression of a branched chain amino-acid transaminase 2, mitochondrial polypeptide (a BCAT2 polypeptide), to decrease expression of a nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 polypeptide (an NFKB1 polypeptide), to decrease expression of a SH2B adaptor protein 1 polypeptide (an SH2B 1 polypeptide), to decrease expression of a lipoprotein lipase polypeptide (an LPL polypeptide), to decrease expression of a 3-hydroxy-3- methylglutaryl-
  • a nutritional supplement composition containing a potato polysaccharide preparation provided herein or a potato polysaccharide preparation provided herein can be used to increase one or more polypeptides (e.g., one or more of an LIPE polypeptide (in adipocytes), a PCK2 polypeptide, an MOGAT1 polypeptide, a PPARGCla polypeptide, a PPARGClb polypeptide, an SOD2 polypeptide, an NR4A1 polypeptide (in adipocytes), an ACAT2 polypeptide, or an HMGCR polypeptide (in muscle cells)) and decrease one or more polypeptides (e.g., one or more of an AGP ATI polypeptide, an OLR1 polypeptide, a BCAT2 polypeptide, an NFKBl polypeptide, an SH2B1 polypeptide, an LPL polypeptide, an HMGCR polypeptide (in adipocytes), an LIPE polypeptide (in muscle cells)
  • R4A1 polypeptide in muscle cells, a PTEN polypeptide, or a CASP8 polypeptide.
  • one aspect of this document features a nutritional supplement composition
  • a nutritional supplement composition comprising, or consisting essentially of, a potato polysaccharide preparation in an amount that, when administered to a mammal, results in between 0.05 mg and 50 mg of the potato polysaccharide component of the potato polysaccharide preparation being administered to the mammal per kg of body weight of the mammal.
  • the composition can comprise between 1 mg and 100 mg of the potato polysaccharide preparation.
  • the composition can comprise between 6 mg and 20 mg of the potato polysaccharide preparation.
  • the composition can comprise between 1 mg and 100 mg of the potato polysaccharide component of the potato polysaccharide preparation.
  • the composition can comprise between 6 mg and 20 mg of the potato polysaccharide component of the potato polysaccharide preparation.
  • the composition can be in the form of a tablet.
  • the composition can comprise alpha lipoic acid.
  • the composition can comprise alpha tocopherol.
  • polysaccharide preparation can be a preparation obtained from raw potatoes.
  • the potato polysaccharide preparation can be in an amount that, when administered to a mammal, results in between 0.075 mg and 0.5 mg of the potato polysaccharide component of the potato polysaccharide preparation being administered to the mammal per kg of body weight of the mammal.
  • At least about 80 percent of the potato polysaccharide preparation can be potato polysaccharide.
  • At least about 90 percent of the potato polysaccharide preparation can be potato polysaccharide.
  • At least about 95 percent of the potato polysaccharide preparation can be potato polysaccharide.
  • the mammal can be a human.
  • this document features a nutritional supplement composition
  • a nutritional supplement composition comprising, or consisting essentially of, a potato polysaccharide preparation obtained from raw potatoes.
  • the composition can comprise between 1 mg and 100 mg of the potato polysaccharide preparation.
  • the composition can comprise between 6 mg and 20 mg of the potato polysaccharide preparation.
  • the composition can comprise between 1 mg and 100 mg of the potato polysaccharide component of the potato polysaccharide preparation.
  • the composition can comprise between 6 mg and 20 mg of the potato polysaccharide component of the potato polysaccharide preparation.
  • the composition can be in the form of a tablet.
  • the composition can comprise alpha lipoic acid.
  • the composition can comprise alpha tocopherol.
  • polysaccharide preparation can be in an amount that, when administered to a mammal, results in between 0.05 mg and 0.5 mg of the potato polysaccharide component of the potato polysaccharide preparation being administered to the mammal per kg of body weight of the mammal.
  • the potato polysaccharide preparation can be in an amount that, when administered to a mammal, results in between 0.075 mg and 0.25 mg of the potato polysaccharide component of the potato polysaccharide preparation being administered to the mammal per kg of body weight of the mammal.
  • the mammal can be a human. At least about 80 percent of the potato polysaccharide preparation can be potato polysaccharide. At least about 90 percent of the potato polysaccharide preparation can be potato polysaccharide. At least about 95 percent of the potato polysaccharide preparation can be potato polysaccharide.
  • this document features a method for increasing polypeptide expression in cells.
  • the method comprises, or consists essentially of, contacting cells with a potato polysaccharide preparation obtained from raw potatoes under conditions wherein expression of one or more of the polypeptides selected from the group consisting of a TFAM polypeptide, an ATP5A1 polypeptide, a PDHA1 polypeptide, a PDHA2 polypeptide, and a THOP1 polypeptide is increased.
  • this document features a method for reducing polypeptide expression in cells.
  • the method comprises, or consists essentially of, contacting cells with a potato polysaccharide preparation obtained from raw potatoes under conditions wherein expression of one or more of the polypeptides selected from the group consisting of a FOX01A polypeptide, a NFKB1 polypeptide, a PDK2 polypeptide, a PDK4 polypeptide, and a HMGCR polypeptide is reduced.
  • this document features a method for treating obesity, diabetes, and/or polycystic ovary syndrome.
  • the method comprises, or consists essentially of, (a) identifying a mammal with obesity, diabetes, and/or polycystic ovary syndrome, and (b) administering to the mammal a potato polysaccharide preparation obtained from raw potatoes.
  • Figure 1 is an HPLC chromatogram of a 10% ACN extract of raw potato (Russet Burbank).
  • Figure 2 is an HPLC chromatogram of collected and re-purified 3.5 minute peak material from a 10% ACN extract of raw potato shown in Figure 1.
  • Figure 3 is a representative real time PCR amplification plot for TFAM expression.
  • Figure 4 is an LC/MS trace of 3.5 minute HPLC peak material.
  • Figure 5 is a full NMR spectrum of 3.5 minute HPLC peak material.
  • Figure 6 is an expanded NMR spectrum of 3.5 minute HPLC peak material.
  • Figure 7 is a total ion chromatogram of derivatized carbohydrate fragments of 3.5 minute HPLC peak material obtained from raw potato Russet Burbank).
  • Figure 8 is a fragmentation pattern of diacetamide.
  • the peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
  • Figure 9 is a fragmentation pattern of 3-acetoxy pyridine.
  • the peak fragmentation pattern is in the top panel, the compound libraiy fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
  • Figure 10 is a fragmentation pattern of 3,4-furan dimethanol, diacetate.
  • the peak fragmentation pattern is in the top panel
  • the compound library fragmentation match is in the bottom panel
  • an overlay of the two is in the center panel.
  • Figure 11 is a fragmentation pattern of 1,2,3-propanetriol diacetate.
  • the peak fragmentation partem is in the top panel
  • the compound libraiy fragmentation match is in the bottom panel
  • an overlay of the two is in the center panel.
  • Figure 12 is a fragmentation pattern of imidazole, 2-acetamino-5-methyl.
  • the peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
  • Figure 13 is a fragmentation pattern of 6,7-dihydro-5H-pyrrol[2,l,c][l,2,4] triazole-3-carboxylic acid.
  • the peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
  • Figure 14 is a fragmentation pattern of acetic acid, l-(2-methyltetrazol-5-yl) ethenyl ester.
  • the peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
  • Figure 15 is a fragmentation pattern of 1,2,3,4-butanetriol, tetraacetate (isomer
  • Figure 1 is a fragmentation pattern of 1,2,3,4-butanetriol, tetraacetate (isomer
  • Figure 17 is a fragmentation pattern of pentaerythritol tetraacetate.
  • the peak fragmentation pattern is in the top panel
  • the compound library fragmentation match is in the bottom panel
  • an overlay of the two is in the center panel.
  • Figure 18 is a fragmentation pattern of 1,2,3,4,5-penta-o-acetyl-D-xylitol (isomer 1).
  • the peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
  • Figure 19 is a fragmentation pattern of 1,2,3,4,5-penta-o-acetyl-D-xylitol (isomer 2).
  • the peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
  • Figure 20 is a fragmentation pattern of 3,5-diacetoxy benzyl alcohol.
  • the peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
  • Figure 21 is a fragmentation pattern of ⁇ -D-galactopyranose, pentaacetate.
  • the peak fragmentation pattern is in the top panel, the compound library
  • fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
  • Figure 22 is a fragmentation pattern of D-mannitol hexaacetate.
  • the peak fragmentation pattern is in the top panel
  • the compound library fragmentation match is in the bottom panel
  • an overlay of the two is in the center panel.
  • Figure 23 is a fragmentation pattern of galacticol, hexaacetate.
  • the peak fragmentation pattern is in the top panel
  • the compound library fragmentation match is in the bottom panel
  • an overlay of the two is in the center panel.
  • Figure 24 is a fragmentation pattern of cyclohexane carboxylic acid, 1,2,4,5- tetrakis(acetoxy), (1 ⁇ ,3 ,4 ⁇ ,5 ⁇ )-(-).
  • the peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
  • Figure 25 is a fragmentation pattern of muco-inositol, hexaacetate.
  • the peak fragmentation pattern is in the top panel
  • the compound library fragmentation match is in the bottom panel
  • an overlay of the two is in the center panel.
  • Figure 26 is a fragmentation pattern of D-glucitol-hexaacetate.
  • the peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
  • Figure 27 is a fragmentation pattern of myo-inositol, hexaacetate.
  • the peak fragmentation pattern is in the top panel
  • the compound library fragmentation match is in the bottom panel
  • an overlay of the two is in the center panel.
  • Figure 28 is an HPLC chromatogram of a 10% ACN extract of raw Organic Yellow potato.
  • Figure 29 is an FtPLC chromatogram of a 10% ACN extract of raw Purple potato.
  • Figure 30 is an HPLC chromatogram of a 10% ACN extract of raw Idaho
  • Figure 31 is an HPLC chromatogram of a 10% ACN extract of raw Yukon Gold potato.
  • Figure 32 is an HPLC chromatogram of a 10% ACN extract of raw sweet potato.
  • Figure 33 is an HPLC chromatogram of a 10% ACN extract of boiled Purple potato.
  • Figure 34 is an HPLC chromatogram of two pooled fraction collections from Idaho Russet potatoes.
  • Figure 35 is an HPLC chromatogram of fractions collections from 3 g of purple potatoes.
  • Figure 36 is an HPLC chromatogram of media collected from cells exposed to a potosaccharide preparation for 4 hours.
  • the document provides nutritional supplement compositions.
  • this document provides nutritional supplement compositions containing a potato polysaccharide preparation, methods for obtaining potato polysaccharide preparations, methods for making nutritional supplement compositions containing a potato polysaccharide preparation, and methods and materials for increasing or decreasing expression of polypeptides involved with mitochondria activity or function.
  • the nutritional supplement compositions provided herein can include one or more potato polysaccharide preparations.
  • a potato polysaccharide preparation can be a preparation that is obtained from a water extract of potato and that contains polysaccharide material having the ability to be eluted from a CI 8 cartridge (e.g., a Sep-Pak Plus C-18 cartridge) with 10% acetonitrile.
  • a potato polysaccharide preparation can be a preparation that is obtained from potato and that contains polysaccharide material having HPLC characteristics of that of the peak eluted at 3.5 minutes as described in Example 1 (see, also, Figures 1, 2, and 28-34).
  • a polysaccharide of a potato polysaccharide preparation provided herein can be a polar, water-soluble polysaccharide. In some cases, a polysaccharide of a potato polysaccharide preparation provided herein can be a highly substituted complex xyloglucan material.
  • a potato polysaccharide preparation can be a preparation that is obtained from potato and that contains polysaccharide material that, when derivatized, results in at least the following acylated carbohydrates as assessed using GC/MS: (a) myo-inositol (set to IX to serve as an internal standard), (b) glucose at about 40X to about 60X the myo-inositol content (e.g., glucose at about 5 OX the myo-inositol content), (c) xylose at about 10X to about 20X the myo-inositol content (e.g., xylose at about 15X the myo-inositol content), (d) mannose at about 5X to about 15X the myo-inositol content (e.g., mannose at about 10X the myo-inositol content), and (e) galactose at about 3X to about 7X the myo-inositol content (e.g.
  • the derivatization procedure can include fonning a dry residue of the polysaccharide material that is then hydro lyzed using trifluoroacetic acid. The resulting material is then reduced using sodium borohydride, and after borate removal, the end product is acylated using acetic anhydride and pyridine. The end products of the reaction are then injected directly on GC/MS to identify the acylated carbohydrates.
  • a potato polysaccharide preparation can be a preparation that is obtained from potato and that contains polysaccharide material that, when derivatized and assessed using GC/MS, results in at least four major components (3,4-furan dimethanol, diacetate; 1,2,3,4,5-penta-o-acetyl-D-xylitol (isomer 1); 3,5-diacetoxy- benzyl alcohol; and D-glucitol-hexaacetate). See, e.g., Example 1.
  • a potato polysaccharide preparation can be a preparation that is obtained from potato and that contains polysaccharide material that, when derivatized and assessed using GC/MS, results in the compounds listed in Table 3 or results in the profile shown in Figure 7.
  • a potato polysaccharide preparation provided herein can be a substantially pure potato polysaccharide preparation.
  • a substantially pure potato polysaccharide preparation is a preparation that contains a single peak of material (e.g., a single peak of polysaccharide material) when assessed using, for example, HPLC (see, e.g., Figures 2 and 34).
  • a single peak of material e.g., a single peak of polysaccharide material
  • HPLC see, e.g., Figures 2 and 34.
  • greater than 60, 70, 75, 80, 85, 90, 95, or 99 percent of a potato polysaccharide preparation provided herein can be polysaccharide material obtained from a potato.
  • Any appropriate potato species or variety can be used to obtain a potato polysaccharide preparation provided herein.
  • Solarium tuberosum, . Ipomoea batatas, S. acaule, S. bukasovii, S. leptophyes, S. megistacrolobum, S. commersonii, or S. infundibuliforme can be used to obtain a potato polysaccharide preparation provided herein.
  • potato varieties of S. tunerosum such as Organic Yellow, Purple or blue varieties, Cream of the Crop, Adirondack Blue, Adirondack Red, Agata, Almond, Andes Gold, Andes Sun, Apline, Alturas,
  • raw potato material can be homogenized (e.g., homogenized with a Polytron homogenizer) in water and maintained at room temperature for a period of time (e.g., about 1 hour) with occasional shaking.
  • the homogenate can be centrifuged (e.g., centrifuged at 4000 g for 10 minutes) to remove any larger solid material.
  • the resulting supernatant can be loaded onto a Solid Phase Extraction cartridge (e.g., a C18 cartridge such as a Sep-Pak Plus C-18 cartridge), and the polysaccharide material eluted with 10 percent acetonitrile. Once eluted, the polysaccharide material can be dried and stored (e.g., stored at about 4°C).
  • This document also provides nutritional supplement compositions containing one or more potato polysaccharide preparations provided herein.
  • a potato polysaccharide preparation provided herein obtained from Idaho Russet potatoes can be formulated into a nutritional supplement composition.
  • Any appropriate dose of a potato polysaccharide preparation provided herein can be used to formulate a nutritional supplement composition.
  • a potato polysaccharide preparation provided herein obtained from Idaho Russet potatoes can be formulated into a nutritional supplement composition.
  • Any appropriate dose of a potato polysaccharide preparation provided herein can be used to formulate a nutritional supplement composition.
  • a potato polysaccharide preparation provided herein obtained from Idaho Russet potatoes can be formulated into a nutritional supplement composition.
  • Any appropriate dose of a potato polysaccharide preparation provided herein can be used to formulate a nutritional supplement composition.
  • a potato polysaccharide preparation provided herein obtained from Idaho Russet potatoes can be formulated into a nutritional supplement composition.
  • polysaccharide preparation provided herein can be used to formulate a nutritional supplement composition such that the nutritional supplement composition contains between about 1 mg and about 750 mg (e.g., between about 1 mg and about 500 mg, between about 1 mg and about 250 mg, between about 5 mg and about 40 mg, between about 5 mg and about 30 mg, between about 5 mg and about 20 mg, between about 6 mg and about 50 mg, between about 6 mg and about 20 mg, between about 10 mg and about 25 mg, or between about 15 mg and about 20 mg) of the potato polysaccharide component of the potato polysaccharide preparation.
  • mg and about 750 mg e.g., between about 1 mg and about 500 mg, between about 1 mg and about 250 mg, between about 5 mg and about 40 mg, between about 5 mg and about 30 mg, between about 5 mg and about 20 mg, between about 6 mg and about 50 mg, between about 6 mg and about 20 mg, between about 10 mg and about 25 mg, or between about 15 mg and about 20 mg
  • a nutritional supplement composition can be formulated to deliver about 0.05 mg of the potato polysaccharide component per kg of body weight to about 0.5 mg of the potato polysaccharide component per kg of body weight to a mammal (e.g., a human) per day.
  • a mammal e.g., a human
  • such nutritional supplement compositions can be formulated into a single oral composition that a human can swallow once a day to provide between about 0.05 mg of the potato polysaccharide component per kg of body weight to about 0.5 mg of the potato polysaccharide component per kg of body weight.
  • a nutritional supplement composition provided herein can be any appropriate method. For example, common formulation mixing and preparation techniques can be used to make a nutritional supplement composition having the components described herein.
  • a nutritional supplement composition provided herein can be in any form.
  • a nutritional supplement composition provided herein can be formulated into a pill, capsule, tablet, gelcap, nutritional shake, nutritional bar, rectal supository, sublingual suppository, nasal spray, inhalant, or injectable ampule.
  • a nutritional supplement composition provided herein can include one or more potato polysaccharide preparations provided herein alone or in combination with other ingredients including, without limitation, gelatin, cellulose, starch, sugar, bentonite, lactic acid, mannitol, alpha lipoic acid, alpha tocopherol, L-ascorbate, or combinations thereof.
  • a potato polysaccharide preparation provided herein or a nutritional supplement composition provided herein can be used to increase or decrease expression of polypeptides involved with mitochondria activity or function.
  • a potato polysaccharide preparation provided herein or a nutritional supplement composition provided herein can be used to increase expression of a TFAM polypeptide, an ATP5A1 polypeptide, a PDHAl polypeptide, a PDHA2 polypeptide, a THOP1 polypeptide, or a combination thereof.
  • a potato polysaccharide preparation provided herein or a nutritional supplement composition provided herein can be used to decrease expression of a FOX01A polypeptide, a NFKB1 polypeptide, a PDK2 polypeptide, a PDK4 polypeptide, a HMGCR polypeptide, or a combination thereof.
  • a potato polysaccharide preparation provided herein or a nutritional supplement composition provided herein can be used to increase one or more polypeptides (e.g., one or more of a TFAM polypeptide, an ATP5A1 polypeptide, a PDHAl polypeptide, a PDHA2 polypeptide, or a THOP1 polypeptide) and decrease one or more polypeptides (e.g., one or more of a FOX01A polypeptide, a NFKB1 polypeptide, a PDK2 polypeptide, a PDK4 polypeptide, or a HMGCR polypeptide).
  • polypeptides e.g., one or more of a TFAM polypeptide, an ATP5A1 polypeptide, a PDHAl polypeptide, a PDHA2 polypeptide, or a THOP1 polypeptide
  • polypeptides e.g., one or more of a FOX01A polypeptide,
  • a potato polysaccharide preparation provided herein or a nutritional supplement composition provided herein can be used to increase one or more human polypeptides (e.g., one or more of a human TFAM polypeptide, a human ATP5A1 polypeptide, a human PDHAl polypeptide, a human PDHA2 polypeptide, a human THOP1 polypeptide, a human LIPE polypeptide (in adipocytes), a human PCK2 polypeptide, a human MOGAT1 polypeptide, a human PPARGCla polypeptide, a vPPARGClb polypeptide, an human SOD2 polypeptide, a human NR4A1 polypeptide (in adipocytes), a human ACAT2 polypeptide, or a human HMGCR polypeptide (in muscle cells)) and/or decrease one or more human polypeptides (e.g., one or more of a human FOX01A polypeptide, a human
  • a human TFAM polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. CAG28581.1 (GI No. 47115243) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM_003201.1 (GI No. 4507400).
  • a human ATP5A1 polypeptide can have the amino acid sequence set forth in GenBank ® Accession No.AAH08028.2 (GI No. 34782901) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession
  • a human PDHA1 polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. ABQ58815.1 (GI No. 148300624) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM_001173454.1 (GI No. 291084741).
  • a human PDHA2 polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. AAH94760.1 (GI No. 66267554) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM_005390.4 (GI No. 134031963).
  • a human THOP1 polypeptide can have the amino acid sequence set forth in GenBank ®
  • a human LIPE polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. AAH70041.1 (GI No. 47124456) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM_005357.2 (GI No. 21328445).
  • a human PCK2 polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. CAG33194.1 (GI No. 48145943) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No.
  • a human MOGAT1 polypeptide can have the amino acid sequence set forth in GenBank Accession No. NP_477513.2 (GI No. 148746191) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM_058165.1 (GI No. 148746190).
  • a human PPARGCla polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. NP_037393.1 (GI No. 7019499) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM O 13261.2 (GI No. 116284374).
  • a human PPARGClb polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. AAI44252.1 (GI No. 219518198) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM_133263.2 (GI No.
  • a human SOD2 polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. AAH16934.1 (GI No. 16877367) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM_000636.1 (GI No. 67782304).
  • a human NR4A1 polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. CAG32985.1 (GI No. 48145525) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No.
  • a human ACAT2 polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. AAH00408.1 (GI No. 12653279) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM_005891.1 (GI No. 148539871).
  • a human FOX01A polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. NP_002006.2 (GI No. 9257222) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM_002015.3 (GI No. 133930787).
  • a human NF B1 polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. CAB94757.1 (GI No. 8574070) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM_001165412.1 (GI No. 25955301).
  • a human PDK2 polypeptide can have the amino acid sequence set forth in GenBank ®
  • NP_002602.2 (GI No. 19923736) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM_00211.4 (GI No.
  • a human PDK4 polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. AAH40239.1 (GI No. 25955471) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM__002612.2 (GI No. 94421466).
  • a human HMGCR polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. AAH33692.1 (GI No.21707182) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM 000859.2 (GI No. 196049378).
  • a human AGP ATI polypeptide can have the amino acid sequence set forth in GenBank ® Accession No.
  • NP_116130.2 (GI No. 15100175) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM_006411.3 (GI No. 301336168).
  • a human OLR1 polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. NP_002534.1 (GI No. 4505501) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. N 002543.2 (GI No. 119392084).
  • a human BCAT2 polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. AAH04243.2 (GI No.
  • a human SH2B1 polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. AAH10704.1 (GI No. 14715079) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM_001145797.1 (GI No. 224926829).
  • a human LPL polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. CAG33335.1 (GI No. 4814622) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No.
  • a human HMGCR polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. AAH33692.1 (GI No. 21707182) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM_001130996.1 (GI No.
  • a human PTEN polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. AAD13528.1 (GI No. 4240387) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No. NM_000314.2 (GI No. 110224474).
  • a human CASP8 polypeptide can have the amino acid sequence set forth in GenBank ® Accession No. AAH68050.1 (GI No. 45751586) and can be encoded by the nucleic acid sequence set forth in GenBank ® Accession No.
  • NM_001228.4 (GI No. 122056470).
  • the potato polysaccharide preparations provided herein or nutritional supplement compositions provided herein can be administered to any mammal (e.g., rat, mouse, dog, cat, horse, cow, goat, pig, chicken, duck, rabbit, sheep, monkey, or human).
  • any route of administration e.g., oral or parenteral
  • a potato polysaccharide preparation provided herein or a nutritional supplement composition provided herein can be administered orally.
  • a potato polysaccharide preparation provided herein or a nutritional supplement composition provided herein can be administered orally.
  • Example 1 Identification of a potato polysaccharide preparation having the ability to alter expression of polypeptides involved with mitochondria activity and function
  • a Waters 2695 separations module with a photodiode array detector was used to purify the 10% ACN extract.
  • An XterraRP C18 column (4.6 X 150 mm) was used for the separation with 0.05% TFA water as the mobile phase.
  • Each HPLC run was a 20 minute gradient ranging from 0 to 2.5% ACN. The injection volume was 100 iL, and the flow rate was 0.5 mL/minute.
  • HPLC fractionation of the 10% ACN extract yielded three major UV absorbing peaks eluted at 3.5, 3.9, and 12.1 minutes ( Figure 1). Collection and HPLC re-purification of the 3.5 minute fraction yielded a symmetrical peak displaying a maximum absorbance at 198.3 nm ( Figure 2).
  • RNA samples were then processed according to the manufacturer's instructions (Qiagen, Valencia, Ca).
  • the RNA was eluted with 40 ⁇ , of RNase-free water by centrifugation for 1 minute at 13,000g.
  • the RNA was analyzed on a model 2100 bioanalyzer (Agilent, Santa Clara, CA) using a total RNA nanochip according to the manufacturer's protocol. Afterwards, 2 ⁇ g of total RNA was reverse transcribed using Superscript III reverse transcriptase and random primers.
  • DNA microarray analyses also were performed using a system provided by Agilent. Arrays included four arrays per chip (Agilent 4X44K chips). Total RNA was reverse transcribed (400 ng) using T7 primers and labeled and transcribed using Cyanine-3 dye. Each array was hybridized with at least 1.65 ⁇ g of labeled cRNA at 65 °C for 18 hours. Arrays were scanned using an Agilent array scanner. A 10% or greater change in gene expression was capable of being determined using both microarray platforms.
  • Real-time PCR was performed in triplicate with TFAM, HMGCR, and NFKB1 detector sets. Beta-actin or GAPDH was used as a reference gene.
  • the realtime PCR master mix included 25 uL 2x universal master mix, 2.5 20x detector set (with the primer and probe), and 21.5 ⁇ , of water.
  • PCR was performed in an Applied Biosystems 7500 sequence detection system.
  • the thermocycler conditions included denaturation at 95°C for 15 seconds and annealing/extension at 60°C for 60 seconds. Forty cycles of PCR were preceded by 95°C for 10 minutes. Reactions were performed in triplicate.
  • the relative quantities of TFAM were found using the formula 2 "AACt using the Applied Biosystems 7500 software.
  • HPLC purified fraction eluting at 3.5 minutes contains polysaccharide material (e.g., highly substituted complex xylogiucan material).
  • polysaccharide material e.g., highly substituted complex xylogiucan material.
  • chromatography/mass spectroscopy (GC/MS), it was converted into its derivatized carbohydrate fragments. Briefly, the sample was concentrated to a dry residue that was hydrolyzed using trifluoroacetic acid. This was then reduced using sodium borohydride, and after borate removal, the end product was acylated using acetic anhydride and pyridine. The end products of the reaction were injected directly on GC MS to identify any acylated carbohydrates. Based on the end analysis, a larger carbohydrate existed in the sample.
  • the total ion chromatogram (TIC) is shown below in Figure 7 with appropriate peak labels below in Table 3. The major components identified are indicated in bold (peaks 3, 12, 14, and 21). The corresponding fragmentation for each compound is provided in Figures 8-27. For each fragmentation, the peak fragmentation pattern is on the top, the compound library fragmentation match is on the bottom, and an overlay of the two is in the center. Finally, unlabeled peaks were either column bleed or did not have a sufficient match to the
  • Example 2 Sweet potatoes and multiple varieties of potatoes exhibit the presence of potosaccharide material
  • Example 3 Highly substituted complex xyloglucan from potato material alters expression of polypeptides in human omental apidocytes obtained from diabetic patients
  • Human omental apidocytes obtained from normal and diabetic patients were purchased from Zen-Bio, Inc (Research Triangle Park, NC). The cells were either untreated or treated with 62.5 ⁇ g/mL of the 3.5 minute peak from purple potatoes for four hours. After the four hour incubations, the cells were harvested, and a microarray analysis was performed to measure changes in gene expression.
  • Real-time PCR was performed in triplicate with AGPAT1, OLRl, BCAT2, NR4A1, and ACAT2 detector sets. Beta-actin or GAPDH was used as a reference gene.
  • the real-time PCR master mix included 25 2x universal master mix, 2.5 20x detector set (with the primer and probe), and 21.5 of water.
  • PCR was performed in an Applied Biosystems 7500 sequence detection system.
  • the thermocycler conditions included denaturation at 95 °C for 15 seconds and annealing/extension at 60°C for 60 seconds. Forty cycles of PCR were preceded by 95°C for 10 minutes. Reactions were performed in triplicate.
  • Example 4 Highly substituted complex xyloglucan from potato material alters expression of polypeptides in mouse myocytes
  • Mouse myoblasts were seeded in 2 niL aliquots into two 75 cm 2 tissue culture flasks. Cells were left to differentiate into myocytes for 4 days in 5% C0 2 at 37 °C.
  • Myocytes were detached from flask walls using gentle agitation. Suspended cells were transferred to a 15mL conical tube and centrifuged at 500g for 3 minutes. 2 mL aliquots were seeded into 75 cm 2 tissue culture flasks for both control and diabetic model cells.
  • the mouse cells were obtained from normal mice and from mice treated with low dose alloxan. The diabetic mice had high blood glucose compared to the normal mice.
  • a potosaccharide preparation (62.5 ⁇ g/mL of the 3.5 minute peak from purple potatoes) was added to one control and one diabetic flask, and the cells were incubated for 24 hours.
  • the cells were harvested, and a microarray analysis was performed to measure changes in gene expression.
  • images were taken of the cells after treatment using a Nikon EclipseTE300 (Morell) inverted microscope coupled with an Optronics digital cameraware at 20x. The images were analyzed on ImageJ software for cell mortality and fiber size.
  • Cell mortality was calculated using a ratio of the number of inactive cells to the number of active cells.
  • Fiber size was calculated using a polygonal lasso tracer and measured in pixel area.
  • Real-time PCR was performed in triplicate with PTEN and CASP8 detector sets. Beta-actin or GAPDH was used as a reference gene.
  • the real-time PCR master mix included 25 ⁇ L 2x universal master mix, 2.5 ⁇ L 20x detector set (with the primer and probe), and 21.5 ⁇ of water.
  • PCR was performed in an Applied Biosystems 7500 sequence detection system.
  • the thermocycler conditions included denaturation at 95°C for 15 seconds and annealing/extension at 60°C for 60 seconds. Forty cycles of PCR were preceded by 95°C for 10 minutes. Reactions were performed in triplicate. Validation of some of the microarray results by real time PCR used PTEN and CASP8 as candidate genes.
  • a potosaccharide preparation was purified using HPLC from 3 g of purple potato.
  • the potosaccharide peak was eluted at about 5 minutes ( Figure 35). This peak was obtained using a different chromatographic column (10 mm xl50 mm) as compared to the column used to obtain the 3.5 minute peak. Since the column was a larger preparative column and the flow rate was 1.5 mL/minute, the elution time of the potosaccharide was 5 minutes.
  • the obtained peak was collected, dried, and reconstituted in 60 ⁇ , of water.
  • the reconstituted potosaccharide material was then added to HTB-11 cells in culture flasks for 4 hours.
  • the post treatment media was collected and added to another flask of HTB-11 cells.
  • Each group of cells was analyzed for gene expression changes.
  • the initially treated cells exhibited the expected changes in mitochondrial gene expression. No changes were detected in the cells exposed to the post treatment media for 4 hours.
  • the post treatment media was extracted using the techniques used to originally purify the potosaccharide.
  • a chromatogram of the extracted post treatment media demonstrated the absence of a peak at 5 minutes.
  • Example 6 - Using a potosaccharide preparation to treat obesity Class I-III obese humans are identified based on the criteria of Table 10. Table 10. Classification of Overweight and Obesity by BMI, Waist Circumference, and Associated Disease Risks.
  • a Class I-III obese patient is treated as follows.
  • Potosaccharide is formulated in the presence of alpha lipoic acid or alpha tocopherol or both.
  • Formulated potosaccharide is added to 90% by weight inert binder material and is administered by the oral parenteral route in the form of a tablet, capsule, or liquid, twice daily (bid).
  • Maximal concentrations of potosaccharide are initially administered bid over the course of one month.
  • Positive outcome measures include: (1) significant reduction of BMI, (2) augmentation of serum LDL/HDL ratio, (3) lowering serum triglyceride concentration, (4) lowering systolic and diastolic blood pressure, and (5) lowering fasting blood glucose.
  • Example 7 Using a potosaccharide preparation to treat type ⁇ diabetes
  • Potosaccharide is formulated in the presence of alpha lipoic acid or alpha tocopherol or both.
  • Formulated potosaccharide is added to 90% by weight inert binder material and is administered by the oral parenteral route in the form of a tablet, capsule, or liquid, twice daily (bid). Maximal concentrations of potosaccharide are initially administered bid over the course of one month.
  • Positive outcome measures include: (1) restoration of normal fasting blood glucose, (2) significant weight loss and lowering of BMI, (3) augmentation of serum LDL/HDL ratio, (4) lowering serum triglyceride concentration, (5) lowering serum concentration of free fatty acids, (6) lowering systolic and diastolic blood pressure, (7) enhancement of insulin sensitivity, and (8) lowering insulin requirement in Type II diabetes patients.
  • POS polycystic ovary syndrome
  • Potosaccharide is formulated in the presence of alpha lipoic acid or alpha tocopherol or both.
  • Formulated potosaccharide is added to 90% by weight inert binder material and is administered by the oral parenteral route in the form of a tablet, capsule, or liquid, twice daily (bid). Maximal concentrations of potosaccharide are initially administered bid over the course of one month.
  • Positive outcome measures include: (1) restoration of normal reproductive function, (2) restoration of normal ovarian follicle maturation, (3) restoration of normal fasting blood glucose levels, (4) significant weight loss and lowering of BMI, (5) augmentation of serum LDL/HDL ratio, (6) lowering serum triglyceride concentration, (7) lowering serum concentration of free fatty acids, (8) lowering systolic and diastolic blood pressure, (9) enhancement of insulin sensitivity, and (10) lowering insulin requirement in comorbid POS patients with type II diabetes.

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Description

METHODS AND MATERIALS RELATED TO NUTRITIONAL
SUPPLEMENT COMPOSITIONS CONTAINING A POTATO POLYSACCHARIDE PREPARATION
BACKGROUND
1. Technical Field
The document relates to nutritional supplement compositions. For example, this document relates to nutritional supplement compositions containing a potato polysaccharide preparation, methods for obtaining potato polysaccharide preparations, methods for making nutritional supplement compositions containing a potato polysaccharide preparation, and methods for increasing or decreasing expression of polypeptides involved with mitochondria activity or function.
2. Background Information
Potatoes are starchy, edible tubers obtained from potato plants and form an integral part of much of the world's food supply. In fact, potatoes are the fourth largest food crop in the world. The main potato species worldwide is Solanum tuberosum.
SUMMARY
The document provides nutritional supplement compositions. For example, this document provides nutritional supplement compositions containing a potato polysaccharide preparation, methods for obtaining potato polysaccharide preparations, methods for making nutritional supplement compositions containing a potato polysaccharide preparation, and methods for increasing or decreasing expression of polypeptides involved with mitochondria activity or function. In some cases, the nutritional supplement compositions and potato polysaccharide preparations provided herein can be used to increase or decrease expression of polypeptides involved with mitochondria activity or function. For example, a nutritional supplement composition containing a potato polysaccharide preparation provided herein or a potato polysaccharide preparation provided herein can be used to increase expression of a transcription factor A, mitochondrial polypeptide (a TFAM polypeptide), an ATP synthase, H+ transporting, mitochondrial Fl complex, alpha subunit 1 polypeptide (an ATP5A1 polypeptide), a pyruvate dehydrogenase (lipoamide) alpha 1 polypeptide (a PDHA1 polypeptide), a pyruvate dehydrogenase (lipoamide) alpha 2 polypeptide (a PDHA2 polypeptide), a thimet oligopeptidase 1 polypeptide (a THOP1 polypeptide), or a combination thereof. In some cases, a nutritional supplement composition containing a potato polysaccharide preparation provided herein or a potato polysaccharide preparation provided herein can be used to decrease expression of a forkhead box 01 polypeptide (a FOXOIA polypeptide), a nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 polypeptide (a NFKBl polypeptide), a pyruvate dehydrogenase kinase, isozyme 2 polypeptide (a PDK2 polypeptide), a pyruvate dehydrogenase kinase, isozyme 4 polypeptide (a PDK4 polypeptide), a 3- hydroxy-3-methylglutaryl-CoA reductase polypeptide (a HMGCR polypeptide), or a combination thereof. In some case, a nutritional supplement composition containing a potato polysaccharide preparation provided herein or a potato polysaccharide preparation provided herein can be used to increase one or more polypeptides (e.g., one or more of a TFAM polypeptide, an ATP5A1 polypeptide, a PDHA1 polypeptide, a PDHA2 polypeptide, or a THOP1 polypeptide) and decrease one or more polypeptides (e.g., one or more of a FOXOIA polypeptide, a NFKBl polypeptide, a PDK2 polypeptide, a PDK4 polypeptide, or a HMGCR polypeptide).
In some cases, the nutritional supplement compositions and potato
polysaccharide preparations provided herein can be used to increase or decrease expression of polypeptides involved with obesity and/or diabetes. For example, a nutritional supplement composition containing a potato polysaccharide preparation provided herein or a potato polysaccharide preparation provided herein can be used to increase expression of a lipase, hormone-sensitive polypeptide (an LIPE polypeptide) in adipocytes, to increase expression of a phosphoenolpyruvate carboxykinase 2 (mitochondrial) polypeptide (a PCK2 polypeptide), to increase expression of a monoacylglycerol O-acyltransferase 1 polypeptide (an MOGAT1 polypeptide), to increase expression of a peroxisome proliferator-activated receptor gamma, coactivator 1 alpha polypeptide (a PPARGCla polypeptide), to increase expression of a peroxisome proliferator-activated receptor gamma, coactivator 1 beta polypeptide (a PPARGClb polypeptide), to increase expression of a superoxide dismutase 2, mitochondrial polypeptide (an SOD2 polypeptide), to increase expression of a nuclear receptor subfamily 4, group A, member 1 polypeptide (an NR4A1 polypeptide) in adipocytes, to increase expression of an acetyl-CoA acetyltransferase 2 polypeptide (an ACAT2 polypeptide), to increase expression of a 3-hydroxy-3-methylglutaryl- CoA reductase polypeptide (an HMGCR polypeptide) in muscle cells, or a combination thereof. In some cases, a nutritional supplement composition containing a potato polysaccharide preparation provided herein or a potato polysaccharide preparation provided herein can be used to decrease expression of a l-acylglycerol-3- phosphate O-acyltransferase 1 polypeptide (an AGP ATI polypeptide), to decrease expression of an oxidized low density lipoprotein (lectin-like) receptor 1 polypeptide (an OLR1 polypeptide), to decrease expression of a branched chain amino-acid transaminase 2, mitochondrial polypeptide (a BCAT2 polypeptide), to decrease expression of a nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 polypeptide (an NFKB1 polypeptide), to decrease expression of a SH2B adaptor protein 1 polypeptide (an SH2B 1 polypeptide), to decrease expression of a lipoprotein lipase polypeptide (an LPL polypeptide), to decrease expression of a 3-hydroxy-3- methylglutaryl-CoA reductase polypeptide (an HMGCR polypeptide) in adipocytes, to decrease expression of a lipase, hormone-sensitive polypeptide (an LIPE polypeptide) in muscle cells, to decrease expression of a nuclear receptor subfamily 4, group A, member 1 polypeptide (an NR4A1 polypeptide) in muscle cells, to decrease expression of a phosphatase and tensin homolog polypeptide (a PTEN polypeptide), to decrease expression of a caspase 8, apoptosis-related cysteine peptidase polypeptide (a CASP8 polypeptide), or a combination thereof.
In some case, a nutritional supplement composition containing a potato polysaccharide preparation provided herein or a potato polysaccharide preparation provided herein can be used to increase one or more polypeptides (e.g., one or more of an LIPE polypeptide (in adipocytes), a PCK2 polypeptide, an MOGAT1 polypeptide, a PPARGCla polypeptide, a PPARGClb polypeptide, an SOD2 polypeptide, an NR4A1 polypeptide (in adipocytes), an ACAT2 polypeptide, or an HMGCR polypeptide (in muscle cells)) and decrease one or more polypeptides (e.g., one or more of an AGP ATI polypeptide, an OLR1 polypeptide, a BCAT2 polypeptide, an NFKBl polypeptide, an SH2B1 polypeptide, an LPL polypeptide, an HMGCR polypeptide (in adipocytes), an LIPE polypeptide (in muscle cells), an
R4A1 polypeptide (in muscle cells), a PTEN polypeptide, or a CASP8 polypeptide).
In general, one aspect of this document features a nutritional supplement composition comprising, or consisting essentially of, a potato polysaccharide preparation in an amount that, when administered to a mammal, results in between 0.05 mg and 50 mg of the potato polysaccharide component of the potato polysaccharide preparation being administered to the mammal per kg of body weight of the mammal. The composition can comprise between 1 mg and 100 mg of the potato polysaccharide preparation. The composition can comprise between 6 mg and 20 mg of the potato polysaccharide preparation. The composition can comprise between 1 mg and 100 mg of the potato polysaccharide component of the potato polysaccharide preparation. The composition can comprise between 6 mg and 20 mg of the potato polysaccharide component of the potato polysaccharide preparation. The composition can be in the form of a tablet. The composition can comprise alpha lipoic acid. The composition can comprise alpha tocopherol. The potato
polysaccharide preparation can be a preparation obtained from raw potatoes. The potato polysaccharide preparation can be in an amount that, when administered to a mammal, results in between 0.075 mg and 0.5 mg of the potato polysaccharide component of the potato polysaccharide preparation being administered to the mammal per kg of body weight of the mammal. At least about 80 percent of the potato polysaccharide preparation can be potato polysaccharide. At least about 90 percent of the potato polysaccharide preparation can be potato polysaccharide. At least about 95 percent of the potato polysaccharide preparation can be potato polysaccharide. The mammal can be a human.
In another aspect, this document features a nutritional supplement composition comprising, or consisting essentially of, a potato polysaccharide preparation obtained from raw potatoes. The composition can comprise between 1 mg and 100 mg of the potato polysaccharide preparation. The composition can comprise between 6 mg and 20 mg of the potato polysaccharide preparation. The composition can comprise between 1 mg and 100 mg of the potato polysaccharide component of the potato polysaccharide preparation. The composition can comprise between 6 mg and 20 mg of the potato polysaccharide component of the potato polysaccharide preparation. The composition can be in the form of a tablet. The composition can comprise alpha lipoic acid. The composition can comprise alpha tocopherol. The potato
polysaccharide preparation can be in an amount that, when administered to a mammal, results in between 0.05 mg and 0.5 mg of the potato polysaccharide component of the potato polysaccharide preparation being administered to the mammal per kg of body weight of the mammal. The potato polysaccharide preparation can be in an amount that, when administered to a mammal, results in between 0.075 mg and 0.25 mg of the potato polysaccharide component of the potato polysaccharide preparation being administered to the mammal per kg of body weight of the mammal. The mammal can be a human. At least about 80 percent of the potato polysaccharide preparation can be potato polysaccharide. At least about 90 percent of the potato polysaccharide preparation can be potato polysaccharide. At least about 95 percent of the potato polysaccharide preparation can be potato polysaccharide.
In another aspect, this document features a method for increasing polypeptide expression in cells. The method comprises, or consists essentially of, contacting cells with a potato polysaccharide preparation obtained from raw potatoes under conditions wherein expression of one or more of the polypeptides selected from the group consisting of a TFAM polypeptide, an ATP5A1 polypeptide, a PDHA1 polypeptide, a PDHA2 polypeptide, and a THOP1 polypeptide is increased.
In another aspect, this document features a method for reducing polypeptide expression in cells. The method comprises, or consists essentially of, contacting cells with a potato polysaccharide preparation obtained from raw potatoes under conditions wherein expression of one or more of the polypeptides selected from the group consisting of a FOX01A polypeptide, a NFKB1 polypeptide, a PDK2 polypeptide, a PDK4 polypeptide, and a HMGCR polypeptide is reduced.
In another aspect, this document features a method for treating obesity, diabetes, and/or polycystic ovary syndrome. The method comprises, or consists essentially of, (a) identifying a mammal with obesity, diabetes, and/or polycystic ovary syndrome, and (b) administering to the mammal a potato polysaccharide preparation obtained from raw potatoes.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. DESCRIPTION OF DRAWINGS
Figure 1 is an HPLC chromatogram of a 10% ACN extract of raw potato (Russet Burbank).
Figure 2 is an HPLC chromatogram of collected and re-purified 3.5 minute peak material from a 10% ACN extract of raw potato shown in Figure 1.
Figure 3 is a representative real time PCR amplification plot for TFAM expression.
Figure 4 is an LC/MS trace of 3.5 minute HPLC peak material.
Figure 5 is a full NMR spectrum of 3.5 minute HPLC peak material.
Figure 6 is an expanded NMR spectrum of 3.5 minute HPLC peak material.
Figure 7 is a total ion chromatogram of derivatized carbohydrate fragments of 3.5 minute HPLC peak material obtained from raw potato Russet Burbank).
Figure 8 is a fragmentation pattern of diacetamide. The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 9 is a fragmentation pattern of 3-acetoxy pyridine. The peak fragmentation pattern is in the top panel, the compound libraiy fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 10 is a fragmentation pattern of 3,4-furan dimethanol, diacetate. The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 11 is a fragmentation pattern of 1,2,3-propanetriol diacetate. The peak fragmentation partem is in the top panel, the compound libraiy fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 12 is a fragmentation pattern of imidazole, 2-acetamino-5-methyl. The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 13 is a fragmentation pattern of 6,7-dihydro-5H-pyrrol[2,l,c][l,2,4] triazole-3-carboxylic acid. The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel. Figure 14 is a fragmentation pattern of acetic acid, l-(2-methyltetrazol-5-yl) ethenyl ester. The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 15 is a fragmentation pattern of 1,2,3,4-butanetriol, tetraacetate (isomer
1) . The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 1 is a fragmentation pattern of 1,2,3,4-butanetriol, tetraacetate (isomer
2) . The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 17 is a fragmentation pattern of pentaerythritol tetraacetate. The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 18 is a fragmentation pattern of 1,2,3,4,5-penta-o-acetyl-D-xylitol (isomer 1). The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 19 is a fragmentation pattern of 1,2,3,4,5-penta-o-acetyl-D-xylitol (isomer 2). The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 20 is a fragmentation pattern of 3,5-diacetoxy benzyl alcohol. The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 21 is a fragmentation pattern of β-D-galactopyranose, pentaacetate. The peak fragmentation pattern is in the top panel, the compound library
fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 22 is a fragmentation pattern of D-mannitol hexaacetate. The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 23 is a fragmentation pattern of galacticol, hexaacetate. The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 24 is a fragmentation pattern of cyclohexane carboxylic acid, 1,2,4,5- tetrakis(acetoxy), (1α,3 ,4α,5β)-(-). The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 25 is a fragmentation pattern of muco-inositol, hexaacetate. The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 26 is a fragmentation pattern of D-glucitol-hexaacetate. The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 27 is a fragmentation pattern of myo-inositol, hexaacetate. The peak fragmentation pattern is in the top panel, the compound library fragmentation match is in the bottom panel, and an overlay of the two is in the center panel.
Figure 28 is an HPLC chromatogram of a 10% ACN extract of raw Organic Yellow potato.
Figure 29 is an FtPLC chromatogram of a 10% ACN extract of raw Purple potato.
Figure 30 is an HPLC chromatogram of a 10% ACN extract of raw Idaho
Russet potato.
Figure 31 is an HPLC chromatogram of a 10% ACN extract of raw Yukon Gold potato.
Figure 32 is an HPLC chromatogram of a 10% ACN extract of raw sweet potato.
Figure 33 is an HPLC chromatogram of a 10% ACN extract of boiled Purple potato.
Figure 34 is an HPLC chromatogram of two pooled fraction collections from Idaho Russet potatoes.
Figure 35 is an HPLC chromatogram of fractions collections from 3 g of purple potatoes.
Figure 36 is an HPLC chromatogram of media collected from cells exposed to a potosaccharide preparation for 4 hours. DETAILED DESCRIPTION
The document provides nutritional supplement compositions. For example, this document provides nutritional supplement compositions containing a potato polysaccharide preparation, methods for obtaining potato polysaccharide preparations, methods for making nutritional supplement compositions containing a potato polysaccharide preparation, and methods and materials for increasing or decreasing expression of polypeptides involved with mitochondria activity or function.
The nutritional supplement compositions provided herein can include one or more potato polysaccharide preparations. A potato polysaccharide preparation can be a preparation that is obtained from a water extract of potato and that contains polysaccharide material having the ability to be eluted from a CI 8 cartridge (e.g., a Sep-Pak Plus C-18 cartridge) with 10% acetonitrile. In some cases, a potato polysaccharide preparation can be a preparation that is obtained from potato and that contains polysaccharide material having HPLC characteristics of that of the peak eluted at 3.5 minutes as described in Example 1 (see, also, Figures 1, 2, and 28-34). In some cases, a polysaccharide of a potato polysaccharide preparation provided herein can be a polar, water-soluble polysaccharide. In some cases, a polysaccharide of a potato polysaccharide preparation provided herein can be a highly substituted complex xyloglucan material.
In some cases, a potato polysaccharide preparation can be a preparation that is obtained from potato and that contains polysaccharide material that, when derivatized, results in at least the following acylated carbohydrates as assessed using GC/MS: (a) myo-inositol (set to IX to serve as an internal standard), (b) glucose at about 40X to about 60X the myo-inositol content (e.g., glucose at about 5 OX the myo-inositol content), (c) xylose at about 10X to about 20X the myo-inositol content (e.g., xylose at about 15X the myo-inositol content), (d) mannose at about 5X to about 15X the myo-inositol content (e.g., mannose at about 10X the myo-inositol content), and (e) galactose at about 3X to about 7X the myo-inositol content (e.g., galactose at about 5X the myo-inositol content). The derivatization procedure can include fonning a dry residue of the polysaccharide material that is then hydro lyzed using trifluoroacetic acid. The resulting material is then reduced using sodium borohydride, and after borate removal, the end product is acylated using acetic anhydride and pyridine. The end products of the reaction are then injected directly on GC/MS to identify the acylated carbohydrates. In some cases, a potato polysaccharide preparation can be a preparation that is obtained from potato and that contains polysaccharide material that, when derivatized and assessed using GC/MS, results in at least four major components (3,4-furan dimethanol, diacetate; 1,2,3,4,5-penta-o-acetyl-D-xylitol (isomer 1); 3,5-diacetoxy- benzyl alcohol; and D-glucitol-hexaacetate). See, e.g., Example 1. In some cases, a potato polysaccharide preparation can be a preparation that is obtained from potato and that contains polysaccharide material that, when derivatized and assessed using GC/MS, results in the compounds listed in Table 3 or results in the profile shown in Figure 7.
In some cases, a potato polysaccharide preparation provided herein can be a substantially pure potato polysaccharide preparation. Typically, a substantially pure potato polysaccharide preparation is a preparation that contains a single peak of material (e.g., a single peak of polysaccharide material) when assessed using, for example, HPLC (see, e.g., Figures 2 and 34). In some cases, greater than 60, 70, 75, 80, 85, 90, 95, or 99 percent of a potato polysaccharide preparation provided herein can be polysaccharide material obtained from a potato.
Any appropriate potato species or variety can be used to obtain a potato polysaccharide preparation provided herein. For example, Solarium tuberosum, . Ipomoea batatas, S. acaule, S. bukasovii, S. leptophyes, S. megistacrolobum, S. commersonii, or S. infundibuliforme can be used to obtain a potato polysaccharide preparation provided herein. In some cases, potato varieties of S. tunerosum such as Organic Yellow, Purple or blue varieties, Cream of the Crop, Adirondack Blue, Adirondack Red, Agata, Almond, Andes Gold, Andes Sun, Apline, Alturas,
Amandine, Annabelle, Anya, Arran Victory, Atlantic, Avalanche, Bamberg, Bamiock Russet, Belle de Fontenay, BF-15, Bildtstar, Bintje, Blazer Russet, Blue Congo,
Bonnotte, British Queens, Cabritas, Camota, Canela Russet, Cara, Carola, Chelina, Chiloe, Cielo, Clavela Blanca, Desiree, Estima, Fianna, Fingerling, Flava, German Butterball, Golden Wonder, Goldrush, Home Guard, Innovator, Irish Cobbler, Jersey Royal, Kennebec, Kerr's Pink, Kestrel, Keuka Gold, King Edward, Kipfler, Lady Balfour, Langlade, Linda, Marcy, Marfona, Maris Piper, Marquis, Megachip, Monalisa, Nicola, Pachacona, Pike, Pink Eye, Pink Fir Apple, Primura, Ranger Russet, Ratte, Record, Red LaSoda, Red Norland, Red Pontiac, Rooster, Russet Burbank, Russet Norkotah, Selma, Shepody, Sieglinde, Silverton Russet, Sirco, Snowden, Spunta, Up to date, Stobrawa, Superior, Vivaldi, Vitelotte, Yellow Finn, or Yukon Gold can be used to obtain a potato polysaccharide preparation provided herein.
Any appropriate method can be used to obtain a potato polysaccharide preparation provided herein. For example, raw potato material can be homogenized (e.g., homogenized with a Polytron homogenizer) in water and maintained at room temperature for a period of time (e.g., about 1 hour) with occasional shaking. The homogenate can be centrifuged (e.g., centrifuged at 4000 g for 10 minutes) to remove any larger solid material. The resulting supernatant can be loaded onto a Solid Phase Extraction cartridge (e.g., a C18 cartridge such as a Sep-Pak Plus C-18 cartridge), and the polysaccharide material eluted with 10 percent acetonitrile. Once eluted, the polysaccharide material can be dried and stored (e.g., stored at about 4°C).
This document also provides nutritional supplement compositions containing one or more potato polysaccharide preparations provided herein. For example, a potato polysaccharide preparation provided herein obtained from Idaho Russet potatoes can be formulated into a nutritional supplement composition. Any appropriate dose of a potato polysaccharide preparation provided herein can be used to formulate a nutritional supplement composition. For example, a potato
polysaccharide preparation provided herein can be used to formulate a nutritional supplement composition such that the nutritional supplement composition contains between about 1 mg and about 750 mg (e.g., between about 1 mg and about 500 mg, between about 1 mg and about 250 mg, between about 5 mg and about 40 mg, between about 5 mg and about 30 mg, between about 5 mg and about 20 mg, between about 6 mg and about 50 mg, between about 6 mg and about 20 mg, between about 10 mg and about 25 mg, or between about 15 mg and about 20 mg) of the potato polysaccharide component of the potato polysaccharide preparation. In some case, a nutritional supplement composition can be formulated to deliver about 0.05 mg of the potato polysaccharide component per kg of body weight to about 0.5 mg of the potato polysaccharide component per kg of body weight to a mammal (e.g., a human) per day. For example, such nutritional supplement compositions can be formulated into a single oral composition that a human can swallow once a day to provide between about 0.05 mg of the potato polysaccharide component per kg of body weight to about 0.5 mg of the potato polysaccharide component per kg of body weight.
Any appropriate method can be used to formulate a nutritional supplement composition provided herein. For example, common formulation mixing and preparation techniques can be used to make a nutritional supplement composition having the components described herein. In addition, a nutritional supplement composition provided herein can be in any form. For example, a nutritional supplement composition provided herein can be formulated into a pill, capsule, tablet, gelcap, nutritional shake, nutritional bar, rectal supository, sublingual suppository, nasal spray, inhalant, or injectable ampule. A nutritional supplement composition provided herein can include one or more potato polysaccharide preparations provided herein alone or in combination with other ingredients including, without limitation, gelatin, cellulose, starch, sugar, bentonite, lactic acid, mannitol, alpha lipoic acid, alpha tocopherol, L-ascorbate, or combinations thereof.
This document also provides methods for increasing or decreasing expression of polypeptides involved with mitochondria activity or function. For example, a potato polysaccharide preparation provided herein or a nutritional supplement composition provided herein can be used to increase or decrease expression of polypeptides involved with mitochondria activity or function. In some cases, a potato polysaccharide preparation provided herein or a nutritional supplement composition provided herein can be used to increase expression of a TFAM polypeptide, an ATP5A1 polypeptide, a PDHAl polypeptide, a PDHA2 polypeptide, a THOP1 polypeptide, or a combination thereof. In some cases, a potato polysaccharide preparation provided herein or a nutritional supplement composition provided herein can be used to decrease expression of a FOX01A polypeptide, a NFKB1 polypeptide, a PDK2 polypeptide, a PDK4 polypeptide, a HMGCR polypeptide, or a combination thereof. In some case, a potato polysaccharide preparation provided herein or a nutritional supplement composition provided herein can be used to increase one or more polypeptides (e.g., one or more of a TFAM polypeptide, an ATP5A1 polypeptide, a PDHAl polypeptide, a PDHA2 polypeptide, or a THOP1 polypeptide) and decrease one or more polypeptides (e.g., one or more of a FOX01A polypeptide, a NFKB1 polypeptide, a PDK2 polypeptide, a PDK4 polypeptide, or a HMGCR polypeptide).
In humans, a potato polysaccharide preparation provided herein or a nutritional supplement composition provided herein can be used to increase one or more human polypeptides (e.g., one or more of a human TFAM polypeptide, a human ATP5A1 polypeptide, a human PDHAl polypeptide, a human PDHA2 polypeptide, a human THOP1 polypeptide, a human LIPE polypeptide (in adipocytes), a human PCK2 polypeptide, a human MOGAT1 polypeptide, a human PPARGCla polypeptide, a vPPARGClb polypeptide, an human SOD2 polypeptide, a human NR4A1 polypeptide (in adipocytes), a human ACAT2 polypeptide, or a human HMGCR polypeptide (in muscle cells)) and/or decrease one or more human polypeptides (e.g., one or more of a human FOX01A polypeptide, a human NFKB1 polypeptide, a human PDK2 polypeptide, a human PDK4 polypeptide, a human HMGCR polypeptide (in adipocytes), a human AGP ATI polypeptide, a human OLRl polypeptide, a human BCAT2 polypeptide, a human SH2B1 polypeptide, a human LPL polypeptide, a human HMGCR polypeptide (in adipocytes), a human LIPE polypeptide (in muscle cells), a human NR4A1 polypeptide (in muscle cells), a human PTEN polypeptide, or a human CASP8 polypeptide).
A human TFAM polypeptide can have the amino acid sequence set forth in GenBank® Accession No. CAG28581.1 (GI No. 47115243) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_003201.1 (GI No. 4507400). A human ATP5A1 polypeptide can have the amino acid sequence set forth in GenBank® Accession No.AAH08028.2 (GI No. 34782901) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession
No.NM_001001937.1 (GI No. 50345983). A human PDHA1 polypeptide can have the amino acid sequence set forth in GenBank® Accession No. ABQ58815.1 (GI No. 148300624) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_001173454.1 (GI No. 291084741). A human PDHA2 polypeptide can have the amino acid sequence set forth in GenBank® Accession No. AAH94760.1 (GI No. 66267554) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_005390.4 (GI No. 134031963). A human THOP1 polypeptide can have the amino acid sequence set forth in GenBank®
Accession No. AAH00583.2 (GI No. 38014202) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_003249.3 (GI No.
34222291). A human LIPE polypeptide can have the amino acid sequence set forth in GenBank® Accession No. AAH70041.1 (GI No. 47124456) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_005357.2 (GI No. 21328445). A human PCK2 polypeptide can have the amino acid sequence set forth in GenBank® Accession No. CAG33194.1 (GI No. 48145943) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No.
NM_004563.1 (GI No. 66346720). A human MOGAT1 polypeptide can have the amino acid sequence set forth in GenBank Accession No. NP_477513.2 (GI No. 148746191) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_058165.1 (GI No. 148746190). A human PPARGCla polypeptide can have the amino acid sequence set forth in GenBank® Accession No. NP_037393.1 (GI No. 7019499) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM O 13261.2 (GI No. 116284374). A human PPARGClb polypeptide can have the amino acid sequence set forth in GenBank® Accession No. AAI44252.1 (GI No. 219518198) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_133263.2 (GI No.
289577087). A human SOD2 polypeptide can have the amino acid sequence set forth in GenBank® Accession No. AAH16934.1 (GI No. 16877367) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_000636.1 (GI No. 67782304). A human NR4A1 polypeptide can have the amino acid sequence set forth in GenBank® Accession No. CAG32985.1 (GI No. 48145525) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No.
NM_173158.1 (GI No. 320202954). A human ACAT2 polypeptide can have the amino acid sequence set forth in GenBank® Accession No. AAH00408.1 (GI No. 12653279) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_005891.1 (GI No. 148539871). A human FOX01A polypeptide can have the amino acid sequence set forth in GenBank® Accession No. NP_002006.2 (GI No. 9257222) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_002015.3 (GI No. 133930787). A human NF B1 polypeptide can have the amino acid sequence set forth in GenBank® Accession No. CAB94757.1 (GI No. 8574070) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_001165412.1 (GI No. 25955301). A human PDK2 polypeptide can have the amino acid sequence set forth in GenBank®
Accession No. NP_002602.2 (GI No. 19923736) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_00211.4 (GI No.
315630394). A human PDK4 polypeptide can have the amino acid sequence set forth in GenBank® Accession No. AAH40239.1 (GI No. 25955471) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM__002612.2 (GI No. 94421466). A human HMGCR polypeptide can have the amino acid sequence set forth in GenBank® Accession No. AAH33692.1 (GI No.21707182) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM 000859.2 (GI No. 196049378). A human AGP ATI polypeptide can have the amino acid sequence set forth in GenBank® Accession No. NP_116130.2 (GI No. 15100175) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_006411.3 (GI No. 301336168). A human OLR1 polypeptide can have the amino acid sequence set forth in GenBank® Accession No. NP_002534.1 (GI No. 4505501) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. N 002543.2 (GI No. 119392084). A human BCAT2 polypeptide can have the amino acid sequence set forth in GenBank® Accession No. AAH04243.2 (GI No. 48257075) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_001190.1 (GI No. 258614013). A human SH2B1 polypeptide can have the amino acid sequence set forth in GenBank® Accession No. AAH10704.1 (GI No. 14715079) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_001145797.1 (GI No. 224926829). A human LPL polypeptide can have the amino acid sequence set forth in GenBank® Accession No. CAG33335.1 (GI No. 4814622) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_000237.1 (GI No. 145275217). A human HMGCR polypeptide can have the amino acid sequence set forth in GenBank® Accession No. AAH33692.1 (GI No. 21707182) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_001130996.1 (GI No.
196049379). A human PTEN polypeptide can have the amino acid sequence set forth in GenBank® Accession No. AAD13528.1 (GI No. 4240387) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_000314.2 (GI No. 110224474). A human CASP8 polypeptide can have the amino acid sequence set forth in GenBank® Accession No. AAH68050.1 (GI No. 45751586) and can be encoded by the nucleic acid sequence set forth in GenBank® Accession No.
NM_001228.4 (GI No. 122056470).
The potato polysaccharide preparations provided herein or nutritional supplement compositions provided herein can be administered to any mammal (e.g., rat, mouse, dog, cat, horse, cow, goat, pig, chicken, duck, rabbit, sheep, monkey, or human). In addition, any route of administration (e.g., oral or parenteral
administration) can be used to administer a potato polysaccharide preparation provided herein or a nutritional supplement composition provided herein to a mammal. For example, a potato polysaccharide preparation provided herein or a nutritional supplement composition provided herein can be administered orally. The document will provide addition description in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1 - Identification of a potato polysaccharide preparation having the ability to alter expression of polypeptides involved with mitochondria activity and function
6 grams of a Russet potato variety of the Solarium tuberosum species were homogenized with a Polytron homogenizer in 20 mL water in a 50 mL centrifuge tube and kept at room temperature for 1 hour. The homogenate was centrifuged at 4000 rpm for 10 minutes. A Sep-Pak Plus C-18 cartridge was activated with 10 mL 100% acetonitrile (ACN) and washed with 10 mL 0.05% trifluoroacetic acid in water (TFA water). 10 mL of the supernatant was loaded onto the cartridge, and all H20 that passes through cartridge was collected in 1.5 mL Eppendorf tubes. Next, 10 mL of 2% ACN (in 0.05%TFA water) was passed through the column, and the elutriate was collected in 1.5 mL Eppendorf tubes. Next, 10 mL of 5% ACN (in 0.05%TFA water) was used to wash the column, and the elutriate was collected in 1.5 mL Eppendorf tubes. Finally, 10 mL of 10% ACN (in 0.05% TFA water) was collected in 1.5 mL Eppendorf tubes after passing through the column. All of the fractions were dried, and the dried fractions of the same ACN concentration were reconstituted into 1 tube in 1 mL of 0.05% TFA water for further purification via HPLC or reconstituted in 1 mL of phosphate buffered saline for use in cell treatments.
A Waters 2695 separations module with a photodiode array detector was used to purify the 10% ACN extract. An XterraRP C18 column (4.6 X 150 mm) was used for the separation with 0.05% TFA water as the mobile phase. Each HPLC run was a 20 minute gradient ranging from 0 to 2.5% ACN. The injection volume was 100 iL, and the flow rate was 0.5 mL/minute. HPLC fractionation of the 10% ACN extract yielded three major UV absorbing peaks eluted at 3.5, 3.9, and 12.1 minutes (Figure 1). Collection and HPLC re-purification of the 3.5 minute fraction yielded a symmetrical peak displaying a maximum absorbance at 198.3 nm (Figure 2).
The three peaks were evaluated to determine whether or not they obtained material having the ability to alter the expression levels of polypeptides involved in mitochondria activity and function. Briefly, 5 x 10s neuroblastoma cells obtained from American Type Culture Collection (ATCC) were plated into each well of 6-well plates with 2 mL of RPMI media and incubated for 4 hours in the presence or absence of different aliquots of the HPLC purified material. Following the incubation, total RNA was isolated and purified using the RNeasy mini kit (Qiagen, Valencia, CA). In particular, pelleted cells were resuspended in 600 μΐΐ of RLT lysis buffer (Qiagen) and homogenized by passing the lysate 20 times through a 1 mL pipette tip. The samples were then processed according to the manufacturer's instructions (Qiagen, Valencia, Ca). In the final step, the RNA was eluted with 40 μΐ, of RNase-free water by centrifugation for 1 minute at 13,000g. The RNA was analyzed on a model 2100 bioanalyzer (Agilent, Santa Clara, CA) using a total RNA nanochip according to the manufacturer's protocol. Afterwards, 2 μg of total RNA was reverse transcribed using Superscript III reverse transcriptase and random primers.
DNA microarray analyses also were performed using a system provided by Agilent. Arrays included four arrays per chip (Agilent 4X44K chips). Total RNA was reverse transcribed (400 ng) using T7 primers and labeled and transcribed using Cyanine-3 dye. Each array was hybridized with at least 1.65 μg of labeled cRNA at 65 °C for 18 hours. Arrays were scanned using an Agilent array scanner. A 10% or greater change in gene expression was capable of being determined using both microarray platforms.
Incubation of cultured cells with the HPLC purified fraction eluted at 3.5 minutes produced changes in the expression of mitochondrial and cellular metabolic genes (Table 1). The extracted potato material that eluted at 3.5 minutes is referred to herein as potosaccharide material or a potosaccharide preparation since it was determined to a polysaccharide as indicated below. The 3.5 minute fraction (a potosaccharide preparation) was the only fraction of the three determined to possess significant biological activity when tested using real time PCR for TFAM, NFKB, and HMGCR expression.
Table 1. Gene expression changes in HTB-11 cells as determined by microarray
Figure imgf000018_0001
PDHA2 pyruvate dehydrogenase (lipoamide) alpha 2 +41
PDK2 pyruvate dehydrogenase kinase, isozyme 2 -24
PDK4 pyruvate dehydrogenase kinase, isozyme 4 -41
HMGCPv 3 -hydroxy-3 -methylglutaryl-CoA reductase -18
THOP1 thimet oligopeptidase 1 +23
Real-time PCR was performed in triplicate with TFAM, HMGCR, and NFKB1 detector sets. Beta-actin or GAPDH was used as a reference gene. The realtime PCR master mix included 25 uL 2x universal master mix, 2.5 20x detector set (with the primer and probe), and 21.5 μΐ, of water. PCR was performed in an Applied Biosystems 7500 sequence detection system. The thermocycler conditions included denaturation at 95°C for 15 seconds and annealing/extension at 60°C for 60 seconds. Forty cycles of PCR were preceded by 95°C for 10 minutes. Reactions were performed in triplicate. The relative quantities of TFAM were found using the formula 2"AACt using the Applied Biosystems 7500 software. Validation of some of the microarray results by real time PCR used TFAM, HMGCR, and NFKB 1 as candidate genes. A representative real time PCR amplification plot demonstrated that TFAM mRNA was present and was differentially expressed (Figure 3). The potosaccharide preparation had a profound effect on TFAM expression and was able to upregulate it by 57% (Table 2). Both HMGCR and NFKB 1 gene expression were reduced by approximately 20%, consistent with and validating the DNA microarray data (Table 2).
Table 2. Validation of gene expression changes by real time PCR. HTB-11 cells treated for 4 hours with a potosaccharide preparation.
Figure imgf000019_0001
Further chemical characterization of the symmetrical 3.5 minute HPLC peak material was performed. Pooled 3.5 minute HPLC fractions were dried and reconstituted in 1 mL TFA water and subjected to tandem LC/MS/MS (Figure 4) and NMR chemical analyses (Figures 5 and 6). For the NMR analysis, Ή-NMR was run on the sample using deuterium oxide (D20) as a solvent to further analyze the sample The water peak at 4.65 PPM was solvent-suppressed, and the spectrum was acquired for several hours. Acetamide was detected at 3.2 PPM, along with acetonitrile at 1.9 PPM. Minor peaks were detected at 1.05 PPM, 1.17 PPM (broad peak), 1.189 PPM, and 1.864 PPM. One characteristic of polymeric materials in a proton NMR was the broadening of peaks such as the shift at 1.17 PPM. These shifts on the NMR could represent the peak at 4.8 PPM and suggested a polar, water-soluble polymer such as a polysaccharide. Taken together, these results confirmed the presence of high molecular weight polysaccharide material contained in HPLC purified fractions eluting at 3.5 minutes.
Further analysis confirmed that the HPLC purified fraction eluting at 3.5 minutes contains polysaccharide material (e.g., highly substituted complex xylogiucan material). To make the polysaccharide material analyzable by gas
chromatography/mass spectroscopy (GC/MS), it was converted into its derivatized carbohydrate fragments. Briefly, the sample was concentrated to a dry residue that was hydrolyzed using trifluoroacetic acid. This was then reduced using sodium borohydride, and after borate removal, the end product was acylated using acetic anhydride and pyridine. The end products of the reaction were injected directly on GC MS to identify any acylated carbohydrates. Based on the end analysis, a larger carbohydrate existed in the sample. The total ion chromatogram (TIC) is shown below in Figure 7 with appropriate peak labels below in Table 3. The major components identified are indicated in bold (peaks 3, 12, 14, and 21). The corresponding fragmentation for each compound is provided in Figures 8-27. For each fragmentation, the peak fragmentation pattern is on the top, the compound library fragmentation match is on the bottom, and an overlay of the two is in the center. Finally, unlabeled peaks were either column bleed or did not have a sufficient match to the compound library.
Figure imgf000020_0001
2013/031700
Figure imgf000021_0001
Figure imgf000022_0001
These results demonstrate the presence of sugar monomers that serve as building blocks for a larger carbohydrate. It appeared from these multiple lines of analysis that the potosaccharide preparation is a highly substituted complex xyloglucan.
Example 2 - Sweet potatoes and multiple varieties of potatoes exhibit the presence of potosaccharide material
Six grams of potato material from multiple varieties of Solarium tuberosum (Organic yellow, Purple, Idaho Russet, and Yukon Gold) and six grams of material from sweet potatoes (Ipomoea batatas) were extracted in 20 mL of water. 10 mL of that water was then loaded onto a sep-pak cartridge, and the cartridge was then eluted with 10 mL of 10% ACN. The ACN was then dried, and the residue was dissolved in 1 mL of water. A 100 uL injection of this water was assessed using HPLC. The HPLC chromatograms demonstrated that the amount of the first peak (at 3.5 minutes at 210 nm) was the same for all five types of potatoes tested (Figures 28- 32).
In another experiment, material was extracted from a boiled Purple potato and analyzed. The peak at 3.5 minutes was not reduced in the boiled potato (Figure 33).
The 3.5 minute peak from two pooled fraction collections from Idaho Russet potatoes was collected, dried, and reconstituted in 100 of water. The material was then injected into the HPLC yielding a single peak at 3.5 minutes (Figure 34). Taken together, these results demonstrate that potatoes within the Solarium tuberosum and Ipomoea batatas species contain potosaccharide material.
Example 3 - Highly substituted complex xyloglucan from potato material alters expression of polypeptides in human omental apidocytes obtained from diabetic patients
Human omental apidocytes obtained from normal and diabetic patients were purchased from Zen-Bio, Inc (Research Triangle Park, NC). The cells were either untreated or treated with 62.5 μg/mL of the 3.5 minute peak from purple potatoes for four hours. After the four hour incubations, the cells were harvested, and a microarray analysis was performed to measure changes in gene expression.
Incubation of human omental apidocytes from diabetic patients with the HPLC purified fraction eluted at 3.5 minutes produced changes in the expression of genes involved in obesity and/or diabetes (Table 4). Incubation of human omental apidocytes from normal humans produced minimal changes in the expression of the genes listed in Table 4 (Table 5).
Table 4. Gene expression changes as determined by microarray following a four-hour incubation of human omental apidocytes from diabetic patients with a potosaccharide preparation.
Figure imgf000023_0001
PCK2 +5
MOGAT1 +52
PPARGCla +59
PPARGClb +44
SOD2 +18
NR4A1 +12
ACAT2 +13
Table 5. Gene expression changes as determined by microarray following a four-hour incubation of human omental apidocytes from normal humans with a potosaccharide preparation.
Figure imgf000024_0001
Real-time PCR was performed in triplicate with AGPAT1, OLRl, BCAT2, NR4A1, and ACAT2 detector sets. Beta-actin or GAPDH was used as a reference gene. The real-time PCR master mix included 25 2x universal master mix, 2.5 20x detector set (with the primer and probe), and 21.5 of water. PCR was performed in an Applied Biosystems 7500 sequence detection system. The thermocycler conditions included denaturation at 95 °C for 15 seconds and annealing/extension at 60°C for 60 seconds. Forty cycles of PCR were preceded by 95°C for 10 minutes. Reactions were performed in triplicate. Validation of some of the microarray results by real time PCR used AGP ATI, OLRl, BCAT2, NR4A1, and ACAT2 as candidate genes. Real time PCR amplification plots demonstrated that AGPAT1, OLRl, BCAT2, NR4A1, and ACAT2 mRNAs were present and were differentially expressed (Table 6). Table 6. Validation of gene expression changes by real time PCR. Human omental apidocytes from diabetic patients treated for 4 hours with a potosaccharide
preparation.
Figure imgf000025_0001
Example 4 - Highly substituted complex xyloglucan from potato material alters expression of polypeptides in mouse myocytes Mouse myoblasts were seeded in 2 niL aliquots into two 75 cm2 tissue culture flasks. Cells were left to differentiate into myocytes for 4 days in 5% C02 at 37 °C.
Myocytes were detached from flask walls using gentle agitation. Suspended cells were transferred to a 15mL conical tube and centrifuged at 500g for 3 minutes. 2 mL aliquots were seeded into 75 cm2 tissue culture flasks for both control and diabetic model cells. The mouse cells were obtained from normal mice and from mice treated with low dose alloxan. The diabetic mice had high blood glucose compared to the normal mice. A potosaccharide preparation (62.5 μg/mL of the 3.5 minute peak from purple potatoes) was added to one control and one diabetic flask, and the cells were incubated for 24 hours.
After the 24 hour incubation, the cells were harvested, and a microarray analysis was performed to measure changes in gene expression. In addition, images were taken of the cells after treatment using a Nikon EclipseTE300 (Morell) inverted microscope coupled with an Optronics digital cameraware at 20x. The images were analyzed on ImageJ software for cell mortality and fiber size. Cell mortality was calculated using a ratio of the number of inactive cells to the number of active cells. Fiber size was calculated using a polygonal lasso tracer and measured in pixel area.
Incubation of mouse myocytes from the diabetic model with the HPLC purified fraction eluted at 3.5 minutes produced changes in the expression of genes involved in obesity and/or diabetes (Table 7). Incubation of mouse myocytes from normal mice produced minimal changes in the expression of the genes listed in Table 7 (Table 8). Table 7. Gene expression changes as determined by microarray following a 24-hour incubation of mouse myocytes from the diabetic model with a potosaccharide preparation.
Figure imgf000026_0001
Table 8. Gene expression changes as determined by microarray following a 24-hour incubation of mouse myocytes from normal mice with a potosaccharide preparation.
Figure imgf000026_0002
Real-time PCR was performed in triplicate with PTEN and CASP8 detector sets. Beta-actin or GAPDH was used as a reference gene. The real-time PCR master mix included 25 \L 2x universal master mix, 2.5 \L 20x detector set (with the primer and probe), and 21.5 μΕ of water. PCR was performed in an Applied Biosystems 7500 sequence detection system. The thermocycler conditions included denaturation at 95°C for 15 seconds and annealing/extension at 60°C for 60 seconds. Forty cycles of PCR were preceded by 95°C for 10 minutes. Reactions were performed in triplicate. Validation of some of the microarray results by real time PCR used PTEN and CASP8 as candidate genes. Real time PCR amplification plots demonstrated that PTEN and CASP8 mRNAs were present and were differentially expressed (Table 9). Table 9. Validation of gene expression changes by real time PCR. Mouse myocytes from the diabetic model treated for 24 hours with a potosaccharide preparation.
Figure imgf000027_0001
A potosaccharide preparation was purified using HPLC from 3 g of purple potato. The potosaccharide peak was eluted at about 5 minutes (Figure 35). This peak was obtained using a different chromatographic column (10 mm xl50 mm) as compared to the column used to obtain the 3.5 minute peak. Since the column was a larger preparative column and the flow rate was 1.5 mL/minute, the elution time of the potosaccharide was 5 minutes.
The obtained peak was collected, dried, and reconstituted in 60 μΐ, of water. The reconstituted potosaccharide material was then added to HTB-11 cells in culture flasks for 4 hours. The post treatment media was collected and added to another flask of HTB-11 cells. Each group of cells was analyzed for gene expression changes. The initially treated cells exhibited the expected changes in mitochondrial gene expression. No changes were detected in the cells exposed to the post treatment media for 4 hours.
In a separate experiment, the post treatment media was extracted using the techniques used to originally purify the potosaccharide. A chromatogram of the extracted post treatment media demonstrated the absence of a peak at 5 minutes.
Example 6 - Using a potosaccharide preparation to treat obesity Class I-III obese humans are identified based on the criteria of Table 10. Table 10. Classification of Overweight and Obesity by BMI, Waist Circumference, and Associated Disease Risks.
Figure imgf000027_0002
18.5-
Normal - - 24.9
25.0-
Overweight Increased High
29.9
30.0-
Obesity I High Very High
34.9
35.0-
II Very High Very High 39.9
Extreme
40.0 + III Extremely High Extremely High Obesity
Once identified, a Class I-III obese patient is treated as follows.
Potosaccharide is formulated in the presence of alpha lipoic acid or alpha tocopherol or both. Formulated potosaccharide is added to 90% by weight inert binder material and is administered by the oral parenteral route in the form of a tablet, capsule, or liquid, twice daily (bid). Maximal concentrations of potosaccharide are initially administered bid over the course of one month. Positive outcome measures include: (1) significant reduction of BMI, (2) augmentation of serum LDL/HDL ratio, (3) lowering serum triglyceride concentration, (4) lowering systolic and diastolic blood pressure, and (5) lowering fasting blood glucose.
Example 7 - Using a potosaccharide preparation to treat type Π diabetes Once a type II diabetes patient is identified, the patient is treated as follows. Potosaccharide is formulated in the presence of alpha lipoic acid or alpha tocopherol or both. Formulated potosaccharide is added to 90% by weight inert binder material and is administered by the oral parenteral route in the form of a tablet, capsule, or liquid, twice daily (bid). Maximal concentrations of potosaccharide are initially administered bid over the course of one month. Positive outcome measures include: (1) restoration of normal fasting blood glucose, (2) significant weight loss and lowering of BMI, (3) augmentation of serum LDL/HDL ratio, (4) lowering serum triglyceride concentration, (5) lowering serum concentration of free fatty acids, (6) lowering systolic and diastolic blood pressure, (7) enhancement of insulin sensitivity, and (8) lowering insulin requirement in Type II diabetes patients. Example 8 - Using a potosaccharide preparation to treat a polycystic ovary syndrome
Once a polycystic ovary syndrome (POS) patient is identified, the patient is treated as follows. Potosaccharide is formulated in the presence of alpha lipoic acid or alpha tocopherol or both. Formulated potosaccharide is added to 90% by weight inert binder material and is administered by the oral parenteral route in the form of a tablet, capsule, or liquid, twice daily (bid). Maximal concentrations of potosaccharide are initially administered bid over the course of one month. Positive outcome measures include: (1) restoration of normal reproductive function, (2) restoration of normal ovarian follicle maturation, (3) restoration of normal fasting blood glucose levels, (4) significant weight loss and lowering of BMI, (5) augmentation of serum LDL/HDL ratio, (6) lowering serum triglyceride concentration, (7) lowering serum concentration of free fatty acids, (8) lowering systolic and diastolic blood pressure, (9) enhancement of insulin sensitivity, and (10) lowering insulin requirement in comorbid POS patients with type II diabetes.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. A nutritional supplement composition comprising a potato polysaccharide preparation in an amount that, when administered to a mammal, results in between 0.05 mg and 50 mg of the potato polysaccharide component of said potato polysaccharide preparation being administered to said mammal per kg of body weight of said mammal.
2. The composition of claim 1, wherein said composition comprises between 1 mg and 100 mg of said potato polysaccharide preparation.
3. The composition of claim 1, wherein said composition comprises between 6 mg and 20 mg of said potato polysaccharide preparation.
4. The composition of claim 1, wherein said composition comprises between 1 mg and 100 mg of the potato polysaccharide component of said potato polysaccharide preparation.
5. The composition of claim 1, wherein said composition comprises between 6 mg and 20 mg of the potato polysaccharide component of said potato polysaccharide preparation.
6. The composition of claim 1, wherein said composition is in the form of a tablet.
7. The composition of claim 1, wherein said composition comprises alpha lipoic acid.
8. The composition of claim 1, wherein said composition comprises alpha tocopherol.
9. The composition of claim 1, wherein said potato polysaccharide preparation is a preparation obtained from raw potatoes.
10. The composition of claim 1, wherein said potato polysaccharide preparation is in an amount that, when administered to a mammal, results in between 0.075 mg and 0.5 mg of the potato polysaccharide component of said potato polysaccharide preparation being administered to said mammal per kg of body weight of said mammal.
11. The composition of claim 1, wherein at least about 80 percent of said potato polysaccharide preparation is potato polysaccharide.
12. The composition of claim 1, wherein at least about 90 percent of said potato polysaccharide preparation is potato polysaccharide.
13. The composition of claim 1, wherein at least about 95 percent of said potato polysaccharide preparation is potato polysaccharide.
14. The composition of claim 1, wherein said mammal is a human.
15. A nutritional supplement composition comprising a potato polysaccharide preparation obtained from raw potatoes.
16. The composition of claim 15, wherein said composition comprises between 1 mg and 100 mg of said potato polysaccharide preparation.
17. The composition of claim 15, wherein said composition comprises between 6 mg and 20 mg of said potato polysaccharide preparation.
18. The composition of claim 15, wherein said composition comprises between 1 mg and 100 mg of the potato polysaccharide component of said potato polysaccharide preparation.
19. The composition of claim 15, wherein said composition comprises between 6 mg and 20 mg of the potato polysaccharide component of said potato polysaccharide preparation.
20. The composition of claim 15, wherein said composition is in the form of a tablet.
21. The composition of claim 15, wherein said composition comprises alpha lipoic acid.
22. The composition of claim 15, wherein said composition comprises alpha tocopherol.
23. The composition of claim 15, wherein said potato polysaccharide preparation is in an amount that, when administered to a mammal, results in between 0.05 mg and 0.5 mg of the potato polysaccharide component of said potato polysaccharide preparation being administered to said mammal per kg of body weight of said mammal.
24. The composition of claim 15, wherein said potato polysaccharide preparation is in an amount that, when administered to a mammal, results in between 0.075 mg and 0.25 mg of the potato polysaccharide component of said potato polysaccharide preparation being administered to said mammal per kg of body weight of said mammal.
25. The composition of claim 23 or 24, wherein said mammal is a human.
26. The composition of claim 15, wherein at least about 80 percent of said potato polysaccharide preparation is potato polysaccharide.
27. The composition of claim 15, wherein at least about 90 percent of said potato polysaccharide preparation is potato polysaccharide.
28. The composition of claim 15, wherein at least about 95 percent of said potato polysaccharide preparation is potato polysaccharide.
29. A method for increasing polypeptide expression in cells, wherein said method comprises contacting cells with a potato polysaccharide preparation obtained from raw potatoes under conditions wherein expression of one or more of the polypeptides selected from the group consisting of a TFAM polypeptide, an ATP5A1 polypeptide, a PDHAl polypeptide, a PDHA2 polypeptide, and a THOPl polypeptide is increased.
30. A method for reducing polypeptide expression in cells, wherein said method comprises contacting cells with a potato polysaccharide preparation obtained from raw potatoes under conditions wherein expression of one or more of the polypeptides selected from the group consisting of a FOXOIA polypeptide, a NFKBl polypeptide, a PDK2 polypeptide, a PDK4 polypeptide, and a HMGCR polypeptide is reduced.
31. A method for treating obesity, diabetes, and/or polycystic ovary syndrome, wherein said method comprises:
(a) identifying a mammal with obesity, diabetes, and/or polycystic ovary syndrome, and
(b) administering to said mammal a potato polysaccharide preparation obtained from raw potatoes.
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