EP1372725A1 - Compositions et methodes pour une fixation non-insulinodependante du glucose - Google Patents

Compositions et methodes pour une fixation non-insulinodependante du glucose

Info

Publication number
EP1372725A1
EP1372725A1 EP02750593A EP02750593A EP1372725A1 EP 1372725 A1 EP1372725 A1 EP 1372725A1 EP 02750593 A EP02750593 A EP 02750593A EP 02750593 A EP02750593 A EP 02750593A EP 1372725 A1 EP1372725 A1 EP 1372725A1
Authority
EP
European Patent Office
Prior art keywords
cell
composition
compound
glucose uptake
concentration
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02750593A
Other languages
German (de)
English (en)
Other versions
EP1372725A4 (fr
Inventor
Dusan VDF Futureceuticals MILJKOVIC
Jovan VDF Futureceuticals HRANISAVLJEVIC
Zbigniew VDF Futureceuticals PIETRZKOWSKI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mitochroma Research Inc
Original Assignee
Mitochroma Research Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2001/007527 external-priority patent/WO2001066146A1/fr
Application filed by Mitochroma Research Inc filed Critical Mitochroma Research Inc
Publication of EP1372725A1 publication Critical patent/EP1372725A1/fr
Publication of EP1372725A4 publication Critical patent/EP1372725A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
    • A61K36/8998—Hordeum (barley)
    • 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

Definitions

  • the field of the invention is dietary supplements and related methods.
  • Elevated blood glucose and blood lipids are a relatively common underlying condition in numerous diseases and may be acquired in various ways.
  • elevated blood glucose levels are frequently precipitated by an altered metabolism associated with a diabetic condition, and treatment of diabetic conditions often includes insulin therapy along with synthetic oral anti-diabetic agents, such as metformin, sulfonylurea, etc.
  • synthetic oral anti-diabetic agents such as metformin, sulfonylurea, etc.
  • various side effects including insulin resistance, allergic reactions, etc. may arise from long- term treatment using insulin.
  • yeast can be grown in the presence of chromium salts, and yeast cells or extracts of cells grown in that manner are particularly rich in "glucose tolerance factor" (GTF), a compound known to enhance the biological effect of insulin.
  • GTF glucose tolerance factor
  • yeast preparations help reduce elevated blood glucose concentrations, in many cases, considerable amounts of yeast preparations must be ingested for a substantial period in order to improve a hyperglycemic condition.
  • long-term use of yeast preparations over extended periods tends to become problematic for some patients, especially in patients who have a history of yeast infections.
  • yeast preparations have a bitter taste that some patients may find objectionable.
  • concentrated, de-bittered and freeze dried yeast preparations have been developed. Such preparations are typically in tablet form, and may conveniently be ingested during a meal. However, the relatively high degree of processing of such cells/extracts may reduce the biological potency of the yeast preparation.
  • preservatives and additives e.g., for pressing or otherwise forming of tablets are typically needed to maintain at least some anti- hyperglycemic activity.
  • chromium picolinate may be administered. Chromium picolinate is reported to be moderately effective in reducing an elevated blood glucose level in human. However, chromium picolinate exhibits considerable toxicity and may therefore not be generally regarded as safe.
  • the present invention is directed to compositions and methods of reducing glucose concentrations in an organism.
  • a composition is isolated from a plant, has AICAR-like activity, and increases glucose uptake into a cell.
  • the composition is preferred to be insulin independent.
  • the composition is also preferred to have a molecular weight Mw of no more than 2000 and a UV light abso ⁇ tion maximum at about 260nm. It is further preferred that the composition is present in a medium surrounding the cell at a concentration of between about 2-10 micrograms per milliliter, and that the medium surrounding the cell is depleted of at least one of a nutrient and oxygen.
  • the cell is contemplated to be a myocyte or part of an in vivo tissue such as liver tissue, skeletal muscle tissue, pancreatic tissue, or adipose tissue.
  • a compound in another aspect of the inventive subject matter, is identical with a molecule isolated from Hordeum vulgare, and has AICAR-like activity, increases glucose uptake into a cell, and the molecule isolated from Hordeum vulgare reduces blood glucose in an organism when the molecule is administered at a concentration effective to reduce the concentration of glucose. It is preferred that the compound is synthesized in vitro and is modified to increase or decrease at least one of the following: an increase in glucose uptake into a cell; solubility in a solvent; chemical stability; and in vivo specificity.
  • a compound is a non-insulin compound that has AICAR-like activity and activates AMPK in a cell. It is preferred that the compound is isolated from a Hordeum vulgare plant seed. It is also preferred that the compound increases glucose uptake in a non-insulin dependent manner when the compound is present in a medium surrounding the cell at a concentration of between 2-10 micrograms per milliliter. It is further preferred that the increase in glucose uptake is reduced when the medium includes L-N-mono-methyl-L-arginine at a concentration of 300 micromoles per liter.
  • a method of treating a cell is contemplated to include the steps of identifying the cell as having a condition that activates AMPK and presenting the cell with a compound having AICAR-like activity at a concentration effective to modulate at least one of an import, export, or synthesis of a molecule.
  • Figure 1 is a flow diagram showing an exemplary method of reducing blood concentration of glucose according to the inventive subject matter.
  • Figure 2 is a flow diagram showing an exemplary method of treating a cell according to the inventive subject matter.
  • Figure 3 is a schematic showing an exemplary preparation of contemplated compounds and thaumatin-like proteins.
  • Figure 4A is a table depicting reduction of blood glucose concentrations in human volunteers using contemplated compositions according to the inventive subject matter.
  • Figure 4B is another table depicting reduction of blood glucose concentrations in human volunteers using contemplated compositions according to the inventive subject matter.
  • Figure 5 A is a table depicting reduction of blood lipid concentrations in human volunteers using contemplated compositions according to the inventive subject matter.
  • Figure 5B is another table depicting reduction of blood lipid concentrations in human volunteers using contemplated compositions according to the inventive subject matter.
  • Figure 6 is a graph depicting fermentation rates of yeast incubated with contemplated compounds at anaerobic and aerobic conditions.
  • Figure 7 is a table depicting intermediate activities by hours of retentate (high molecular weight) and permeate (low molecular weight) of GMM sample after dialysis.
  • Figure 8 is a graph depicting fractions of eluates from the retentate portion of GMM sample.
  • Figure 9 is another graph depicting fractions of eluates from the retentate portion of GMM sample.
  • Figure 10 is another graph depicting fractions of eluates from the retentate portion of
  • Figure 11 is a graph depicting L6 cells treated with insulin in serum-free and serum- supplemented medium.
  • Figure 12 is a graph depicting the effect of the permeate portion of GMM sample on glucose uptake in L6 cells.
  • Figure 13 is a graph depicting the effect of AICA, L-NMMA, and AICAR + L- NMMA on glucose uptake.
  • Figure 14 is a graph depicting the effect of the permeate portion of GMM sample and the effect of the permeate portion + L-NMMA on glucose uptake.
  • a method 100 of reducing a glucose concentration in an organism has a step 110 in which a composition is provided that includes a compound that binds to a thaumatin-like protein.
  • the composition is administered to the mammal in a dosage effective to decrease the blood concentration of glucose.
  • the term "compound that binds to a thaumatin-like protein” refers to any compound or mixture of compounds that exhibit a binding preference to a thaumatin- like protein from barley of at least 10-fold, more preferably at least 100-fold over binding to other barley proteins, wherein binding of contemplated compounds to the thaumatin-like protein will preferably have a KQ of less than 10 "3 M, more preferably of less than 10 " *M.
  • the mode of binding need not be limited to a single interaction (e.g., hydrophobic interaction), but may include multiple interactions (e.g., electrostatic interactions and hydrogen bonding, etc.). It is especially contemplated that binding is reversible, however, irreversible binding is not excluded.
  • thaumatin-like proteins from barley are generally preferred binding partners for compounds according to the inventive subject matter
  • thaumatin-like proteins from alternative sources including microorganisms, plants, and animals are also contemplated.
  • Thaumatin-like proteins are a well characterized class of polypeptides and are described, for example, in Cvetkovic et al, J. Serb Chem. Soc. 62(9):777-786 (1997), Cvetkovic et al, J. Serb. Chem. Soc. 62(l):51-56 (1997) and Cvetkovic et al., J. Inst. Brew. 103:183-186 (1997), all of which are incorporated by reference herein.
  • a method 100 of treating a cell has a step 110 in which the cell has a condition that activates an AMPK (adenosine 5'-monophosphate-activated protein kinase).
  • the cell is presented with a compound having AICAR-like activity at a concentration effective to modulate at least one of an import, export, and synthesis of a molecule.
  • AICAR refers to 5'-aminoimidazole 4-carboxamide 1-ribonucleotide.
  • AICAR-like activity and “5'-aminoimidazole 4-carboxamide 1- ribonucleotide” refer to any activity that stimulates insulin independent glucose uptake into L-6 muscle cells that is inhibited by L-NMMA, and which does not include AICA or AICAR or glucose. Also, contemplated compounds do not include tocol or tocotriene compounds.
  • L-NMMA refers to NG-methyl-L-arginine, acetate salt, which is an inhibitor of nitric oxide synthase (NOS).
  • AMPK has known metabolic regulatory effects on fatty acid and carbohydrate metabolism in liver, adipose tissue, pancreatic beta-cells, and skeletal muscle. AMPK is known to effect increases in glucose uptake and increases in fatty acid oxidation in skeletal muscle, increases in fatty acid oxidation, decreases in cholesterol synthesis, and decreases in lipogenesis in the liver. Furthermore, AMPK is known to modulate insulin secretion in pancreatic islets.
  • the composition is a pharmaceutical or nutraceutical for treatment of diabetes.
  • the composition is prepared from Hordeum vulgare (as outlined in examples, infra), and orally administered in 3 daily doses of 500mg, respectively, to a human diagnosed with non-insulin dependent diabetes mellitus (NIDDM).
  • NIDDM non-insulin dependent diabetes mellitus
  • especially preferred compositions include a compound that binds to thaumatin-like proteins and that reduces a concentration of glucose in an organism when the compound is administered to the organism at a concentration effective to reduce the concentration of glucose.
  • compositions and compounds need not be limited to a preparation from Hordeum vulgare, but may also include preparations from various plants other than Hordeum vulgare, and particularly contemplated alternative plants include Hordeum spec, and members of the poaceae family. While the preparation of contemplated compositions and/or compounds is preferably from plant extracts, it should further be appreciated that contemplated compositions and/or compounds may also be isolated from microorganisms (i.e., bacteria, fungi, yeasts, unicellular eucaryotic organisms) or animals, so long as contemplated compounds bind to a thaumatin-like protein and reduce a glucose concentration in an organism.
  • microorganisms i.e., bacteria, fungi, yeasts, unicellular eucaryotic organisms
  • contemplated compositions are identical with, or a derivative of, a molecule isolated from Hordeum vulgare.
  • Derivatives can be advantageously developed to address issues of bioavailability, targeting and other specificity, efficacy, and reduction in toxicity.
  • the molecule may be isolated from Hordeum vulgare through any procedure including malting, mashing, salt extraction, a buffer extraction, ethanol extraction, anion exchange chromatography, and molecular sieving. Still further, it is contemplated that the molecule is isolated from any part of the Hordeum vulgare or a plant seed of Hordeum vulgare. It is preferred that the molecule is isolated from a malted seed of Hordeum vulgare.
  • contemplated compounds may be isolated, purified to homogeneity, and the structure be elucidated. Consequently, it should be appreciated that contemplated compounds and/or compositions may be entirely (de novo) or partially synthesized/modified in vitro.
  • a precursor of contemplated compounds may be isolated from a plant or microorganism, and then be subjected to one or more steps to arrive at contemplated compounds.
  • contemplated compounds may be modified in one or more synthetic steps to impart a particularly desirable physico-chemical property.
  • contemplated compounds may be esterified with a polar compound (e.g, polyethylene glycol) to increase water solubility.
  • contemplated compounds may be coupled to a resin or other material to control the rate of release to the organism.
  • Preferred contemplated compounds have a relatively low molecular weight, typically no more than 2000Da, however, it should be recognized that the molecular weight may vary considerably and will predominantly depend on the source from which the compound is isolated, synthetic modifications, dimerizations and multimerizations. Likewise, it is contemplated that suitable compounds need not be limited to compounds having a UV abso ⁇ tion maximum at about 260nm (which is characteristic for contemplated compounds isolated using the procedure outlined below), and various spectral characteristics other than a UN 260 peak are also suitable.
  • Hpophilic solvent includes all solvents that have a miscibility with H 2 O of less than 10vol%.
  • contemplated compounds are chemically substantially pure (i.e., concentration of contemplated compounds greater than 90wt%, preferably greater than 95wt%, most preferably greater than 99wt%)
  • contemplated compounds may be coupled to one or more than one molecule, and particularly contemplated molecules include thaumatin-like proteins.
  • contemplated compositions include complexes between contemplated compounds and thaumatin-like proteins, and especially include complexes between contemplated compounds and thaumatin-like proteins as they are isolated from the appropriate sources (infra).
  • glucose concentration is a blood glucose concentration.
  • glucose concentrations also include concentrations of glucose covalently or non-covalently bound to molecules found within the organism, and especially contemplated alternative glucose concentrations include concentrations of glycosylated proteins (e.g., glycosylated hemoglobin or collagen).
  • thaumatin-like proteins are isolated from Hordeum vulgare
  • alternative thaumatin-like proteins are also contemplated and include thaumatin-like proteins isolated from microorganisms, plants, and animals, which may or may not be expressed in a recombinant system.
  • thaumatin-like proteins isolated from microorganisms, plants, and animals, which may or may not be expressed in a recombinant system.
  • There are various protocols for isolation of thaumatin-like proteins known in the art see e.g., Barre et al, Purification and structural analysis of an abundant thaumatin-like protein from ripe banana fruit. Planta.
  • contemplated compositions not only reduce elevated blood glucose concentration in human suffering from NIDDM, but may also reduce blood glucose concentrations in individuals having elevated blood glucose concentrations for reasons other than NIDDM, including obesity, dietary effects, etc. It is especially contemplated that individuals with or without NIDDM will have a blood glucose concentration of at least 90mg/dl, more preferably of at least 120 mg/dl, and most preferably of at least 200 mg/dl.
  • compositions have also been shown to advantageously reduce elevated blood lipid concentrations (infra), wherein blood lipids particularly include triglycerides, fatty acids, HDL-cholesterol, and LDL-cholesterol, and it is further contemplated that the reduction of blood lipids may be concomitantly with the reduction of blood glucose levels, or independent of the reduction of the blood glucose level.
  • blood lipids particularly include triglycerides, fatty acids, HDL-cholesterol, and LDL-cholesterol
  • the reduction of blood lipids may be concomitantly with the reduction of blood glucose levels, or independent of the reduction of the blood glucose level.
  • compositions may further comprise active or inactive ingredients, including compositions known to decrease a blood lipid concentration, and/or compositions known to decrease blood sugar concentrations.
  • alternative compositions may include at least one of a tocol, vitamins, and/or mineral preparations, GTF, metformin, sulfonylurea, and the like.
  • Inactive ingredients include fillers, coloring agents, stabilizers, and the like.
  • an exemplary method of treating a person having an increased blood concentration of glucose of approximately 150 mg/dl, and an increased blood concentration of total cholesterol of above 280 mg/dl, or more has one step in which contemplated compositions are provided.
  • the composition is administered to the person in a dosage effective to decrease the concentration of glucose.
  • compositions according to the inventive subject matter are non-GTF compositions.
  • the duration for contemplated treatments may vary significantly, and suitable durations may be within the range of a single dose, but also for a predetermined period, including one week, several weeks, several months, and even several years. Consequently, it should be appreciated that compositions according to the inventive subject matter may also be prophylactically administered to a human to prevent hyperglycemia, or some form of dyslipidemia.
  • contemplated compositions increase glucose uptake into a cell.
  • the cell may be any cell including a myocyte, or part of a tissue. It is further contemplated that the cell may be insulin dependent or insulin independent.
  • Contemplated tissues are preferred to be in vivo, and may include liver tissue, skeletal muscle tissue, pancreatic tissue, and adipose tissue. It is further preferred that the cell is a myocyte and is insulin independent.
  • Contemplated compositions may be present in a medium surrounding the cell, such as the serum or interstitial fluids. It is further contemplated that the composition may increase glucose uptake into a cell when the compound is present in a medium surrounding the cell at a concentration of between 2 -10 micrograms per milliliter. Furthermore, it is contemplated that the cell or the medium surrounding the cell is depleted of at least one of a nutrient and oxygen. The term “depleted” refers to less than 10% of a normally fit cell or less than 15% of a particular nutrient (i.e. glucose).
  • an increase in glucose uptake is reduced when the medium surrounding the cell comprises L-N-mono-methyl-L-arginine at a concentration of 300 micromoles per liter. It is further contemplated that the composition may be modified to increase or decrease an increase of glucose uptake into a cell. Furthermore, it is contemplated that the composition may be modified to increase or decrease solubility in a solvent, chemical stability, or in vivo specificity.
  • composition may also be administered to an organism other than a human, and particularly preferred alternative organisms include livestock (e.g., cattle, pigs, horses, etc.) and pets (e.g, dogs, cats, rodents, birds, etc.).
  • livestock e.g., cattle, pigs, horses, etc.
  • pets e.g, dogs, cats, rodents, birds, etc.
  • treatment according to the inventive subject matter may also result in significant weight loss, particularly in persons with obesity, NIDDM, or other condition associated with increased body weight.
  • the treatment according to the inventive subject matter is not limited to reduction of blood glucose alone, but may concomitantly (or by itself) include reduction of a particular lipid or lipid group.
  • slightly elevated total cholesterol e.g., 220 mg/dl
  • an imbalance between HDL and LDL i.e. LDL»HDL
  • treatment with the contemplated method may still be indicated due to an elevated triglyceride level.
  • oral preparations there are many alternative oral preparations besides 3 oral daily doses of 500mg.
  • dosages may increase from 500mg - 5g per day, and more.
  • High dosages may also be required where the potency of an extract is relatively low.
  • low dosages e.g., maintenance therapy
  • daily dosages between 500mg and 25mg, or less, are appropriate. Therefore, it is generally contemplated that among other parameters the patient's particular condition and the potency of the preparation will at least partially determine the frequency of application.
  • oral administration need not be limited to a tablet, and alternative oral administrations may include powders, gel-caps, syrups, gels, etc. Where oral administration is not desirable, it is further contemplated that alternative routes are also appropriate, including injections, transdermal, pulmonary or intranasal delivery.
  • Example 1 and 2 describe basic and improved procedures of producing compositions according to the inventive subject matter, respectively.
  • the biological activity of the compounds isolated according to procedures in Examples 1 and 2 is described in Example 3 and 4, and Example 5 provides experimental support for specific binding of contemplated compounds to thaumatin-like proteins.
  • Example 6 provides experimental procedures to determine the molecular weight of molecules in GMM.
  • Example 7 provides experimental data for chromatographic fractions of GMM retentate, and example 8 provides experimental support for an increase of glucose uptake in L6 muscle cells of GMM permeate.
  • Barley grains were malted according to procedures well known in the art of beer brewing (see e.g., Principles of Brewing Science, Second Edition, by George J. Fix; Brewers Publications; ISBN: 0937381748, or The Brewers' Handbook by Ted Goldhammer; KVP Publishers; ISBN: 0967521203).
  • a step of mashing was subsequently applied to the ground malt (suspended in water) according to a typical brewer's schedule.
  • the temperature cycles were as follows: Incubation at 40°C for 60min, incubation at 50°C for 60 min, incubation at 60°C for 60 min, incubation at 72°C for 60 min, and incubation at 75°-80°C for 60 min. Soluble portions of samples were separated from husks and other insoluble material and freeze-dried.
  • the freeze-dried barley extract obtained after mashing at 40°C served as base for fractionation into its components.
  • a first fractionation was achieved by preparative liquid chromatography using a DEAE-Sephacel column (2.6 x 20 cm) equilibrated with 50mM phosphate buffer, pH 7.8. 150 mg of the freeze-dried sample was dissolved in 10 ml of buffer and placed on the column. A linear NaCl-gradient (0 - 0.5 M) was run at a flow rate of 10 ml/h. Fractions (2 ml each) were collected, and elution was monitored at 280 nm.
  • the DEAE chromatography resulted in four distinct protein peak fractions: I - basic, II - neutral, III- and IV - acidic.
  • Respective peak fractions were collected, desalted and concentrated by membrane ultra-filtration using a membrane cut-off pore size of 1000 Dalton, and concentrated corresponding fractions were checked for their capacity to influence yeast fermentation rate.
  • the basic fraction I produced significant inhibitory effect (i.e., a reduction of the yeast fermentation rate), while the remaining three concentrated fractions were almost inert.
  • the main proteinaceous component in fraction I represent thaumatin-like proteins. It has been noticed during the membrane ultra- filtration of the pooled protein fractions I - IV (i.e., fractions obtained by ion exchange chromatography), that the filtrate of some fractions contains LMW (low molecular weight) substances with a UV absorbance maximum of approximately 260 nm. These observations prompted us to employ molecular sieving chromatography to separate these LMW substances from proteins in these fractions.
  • LMW low molecular weight
  • GMM-1 GMM- 1
  • GMM- 2 GMM-4
  • LMW LMW components
  • All of GMM- 1, GMM- 2 and GMM-4 enhanced yeast fermentation, bound strongly and reversibly to thaumatin-like protein (bind to thaumatin-like proteins at low salt condition and release from thaumatin-like proteins at high salt condition), and reduced elevated blood glucose concentration and elevated blood lipid concentration in human diagnosed with NIDDM.
  • aqueous extract was separated from the suspension by vacuum filtration over a cellulose filter pad.
  • citrate or other buffers are also contemplated suitable for preparation of an aqueous extract.
  • the filtered extract was freeze-dried or vacuum-evaporated. So obtained dry malt extract (yield approx. 12-14 g) contained 5.6 g of NaCl originating from the extracting solvent and a complex mixture of water-soluble barley components.
  • the filtered freeze- dried extract was purified by extraction with two 50 ml portions of warm ethanol under vigorous mixing for two hours. The ethanolic extracts were filtered, combined, and evaporated to an oily residue in vacuum. The oily residue was re-dissolved in 15 ml of water and freeze-dried, resulting in a hard glassy yellowish product in a total amount of approx. 3 g-
  • the glassy yellowish product enhanced yeast fermentation, bound strongly and reversibly to thaumatin-like protein (bind to thaumatin-like proteins at low salt condition and release from thaumatin-like proteins at high salt condition), and reduced elevated blood glucose concentration and elevated blood lipid concentration in human diagnosed with NIDDM.
  • compositions comprise a plant seed extract (preferably from Hordeum vulgare), wherein the plant seed is malted (preferably at a temperature between about 30°C and 65°C) and the extract is prepared from the malted plant seed using a protocol that includes an aqueous extraction step (e.g., using an aqueous buffer such as a citrate buffer), and that the extract reduces a glucose concentration in an organism when the extract is administered to the organism at a concentration effective to reduce the concentration of glucose.
  • a plant seed extract preferably from Hordeum vulgare
  • the extract is prepared from the malted plant seed using a protocol that includes an aqueous extraction step (e.g., using an aqueous buffer such as a citrate buffer), and that the extract reduces a glucose concentration in an organism when the extract is administered to the organism at a concentration effective to reduce the concentration of glucose.
  • yeast cells Two grams of wet brewers yeast cells (about 20% dry weight) were suspended in fermentation medium (25 ml of 60 mM phosphate buffer, pH 5.7 and 10 ml of 5% (w/v) glucose solution), and aliquots of the products from example 1 or 2 were added to the fermentation medium for testing. Incubations were carried out in 50ml fermentation flasks at 25°C for 60 minutes. The fermentation rates were measured from the volume of generated CO 2 . All of the tested LMW fractions or the product from Example 2 showed significant biological activity or bioactivity in that they increased the yeast fermentation rate in the range of about 20 - 40%. As used herein, a bioactive compound is one that increases or decreases fermentation. In a further experiment, the activity of GMM-2 was checked at aerobic conditions.
  • Example 2 The product obtained in Example 2 was examined for use in humans diagnosed with
  • NIDDM Newcastle disease virus
  • 25 men were recruited from an outpatient clinic (Endocrinology Department). Mean age within the group was 51 yr, ranging from 36 to 74. Medical records were screened to exclude diabetics taking insulin or oral hypoglycemic agents. All of the subjects agreed to maintain their usual eating habits and health-related behaviors throughout the study. The experimental treatments were run over a period of six month. The participants were instructed to take the preparation in 3 oral daily doses of 1,000 mg each in a tablet form.
  • Plasma glucose According to the plasma glucose levels the subjects were subdivided in three groups for differentiation of the effects: I - up to 8 mMol/L; II - 8 - 10.5 mMol/L and III - above 10.5 mMol L of plasma glucose concentration.
  • Glycosylated hemoglobin (HbAcl) According to the HbAcl levels the subjects were divided in two groups: I - below 10% and II - above 10% of the modified hemoglobin. The test results related to glycemia, before and after treatment, are shown in Figure 4A.
  • the lipid status of the subjects diagnosed with NIDDM was determined before and after treatment by testing plasma level of triglycerides, and cholesterol (as total, LDL and HDL form).
  • the test results shown in Figures 5A and 5B include subjects with disturbed lipid metabolism due to diabetic disease.
  • the lipid status of the subjects as shown in Figure 5 A includes plasma levels of triglycerides, the ratio of triglycerides over total cholesterol, and the ratio of LDL/HDL. The latter two ratios are known as atherosclerotic risk factors.
  • administration of contemplated compounds resulted in a reduction of triglycerides of up to 50%, and a significant reduction of about 1-20% of the ratio of triglycerides to HDL cholesterol, with an even more dramatic reduction of the ratio between LDL to HDL cholesterol (about 40%).
  • the lipid status as shown in Figure 5B includes further results of ten test patients after administration of contemplated compounds and/or compositions over a period of 90 days.
  • Thaumatin-like proteins were prepared following the procedure as generally outlined in Example 1 and Figure 3. So isolated thaumatin-like proteins were subjected to repeated molecular sieving in a membrane concentrator using a membrane with a molecular weight cut off of about lOOODalton. After a first round of filtration of the protein preparation, 99ml of buffer (50 mM phosphate buffer, pH 7.8, 0,5 M NaCl) were added to about 1ml of retentate (i.e. the thaumatin-like protein fraction), and three subsequent rounds of filtration were performed with the same buffer to remove remaining GMM-compounds (i.e., herein presented compounds that reduce elevated glucose) from the thaumatin-like protein preparation.
  • buffer 50 mM phosphate buffer, pH 7.8, 0,5 M NaCl
  • UV absorbance of the filtrate was monitored at 260nm and the biological activity of sample volumes from the filtrate was tested according to protocols outlined in Example 3.
  • Such prepared thaumatin-like proteins were desalted by membrane filtration employing NaCl-free buffer (50 mM phosphate buffer, pH 7.8), and further used in the following procedure:
  • a desalted thaumatin-like protein solution (10 mg/ml)
  • 1.0 ml of a GMM-1 solution (1 mg/ml) was added, and the mixture was incubated at room temperature for 2hrs. After 2 hrs, 98 ml of 50 mM phosphate buffer, pH 7.8 were added to the mixture and unbound GMM-1 was removed by 3 subsequent rounds of ultrafiltration (each round 1:100 by volume) with buffer.
  • Sample 1 The thaumatin-like protein with the bound GMM-1 was labeled Sample 1.
  • Sample 1 was then subjected to a molecular sieving chromatography using a Sephadex G-75 column with 50 mM phosphate buffer, pH 7.8, 0.5 M NaCl as solvent, in which a low molecular weight fraction eluted with an absorbance of 260nm separate from a higher molecular weight fraction of the thaumatin-like protein with absorbance of 280nm.
  • the low molecular weight fraction was concentrated, desalted, and brought to a volume of 1.0ml and labeled Sample 2.
  • Samples 1 and 2 were then tested for biological activity employing a procedure as outlined in Example 3.
  • compositions and methods to reduce glucose concentrations in an organism have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended contemplated claims. Moreover, in inte ⁇ reting both the specification and the contemplated claims, all terms should be inte ⁇ reted in the broadest possible manner consistent with the context.
  • GMM was dialyzed in order to determine the molecular weight distribution of active GMM components and to purify and concentrate the active components.
  • a series of experiments was performed in portions of 3 g of GMM preparation per one tube. GMM samples were dissolved in 40 ml of water and dialyzed against 500 ml of water for 24 hours. External water was replaced with fresh water after 24 hours, for better separation. The external water portions were combined, concentrated in a vacuum, centrifuged, and . evaporated to dryness, resulting in permeate. The contents of the dialysis tubes were centrifuged and evaporated to dryness in a vacuum, resulting in retentate.
  • the percentage ratio of permeate to retentate was 58.5: 25.1, with 16.4 corresponding to insoluble materials from both retentate and permeate. This insoluble part was discarded.
  • the kinetics (rate of action) of the two soluble fractions in yeast fermentation is shown in Figure 7.
  • Figure 7 shows that at least two types of active compounds in GMM are present. These two types include: high molecular weight (HMW, retentate) substance(s) with an increase of activity with the progress of the fermentation process, and low molecular weight (LMW, permeate) substance(s) with an early activity decreasing with progress of fermentation.
  • HMW high molecular weight
  • LMW low molecular weight
  • permeate kinetic is overlapped with retentate activity.
  • the retentate activity results in a significant increase of yeast biomass in a range of 44% over the control.
  • the results may indicate that the retentate GMM fraction acts at a gene level leading to cell multiplication, while the permeate GMM fraction most probably exerts its action through an enhanced glucose uptake in a non-insulin dependent pathway.
  • the retentate sample from Example 6 was subject to gel permeation chromatography with Sephadex G-75-50 at column of 2 x 80 cm using 50 mM phosphate buffer, pH 7.8.
  • the GMM/R samples were 40 - 50 mg dissolved in the buffer.
  • the eluates were fractionated in the fraction collector in 15 minutes intervals, giving 1.5 ml fractions. The elution was traced by spectrophotometric measurement at and absorbed at 280 nm.
  • Yeast activity tests were then run using Barometric methods, which were more convenient than Gravimetric methods. The tests were run parallel and simultaneous to the control. The eluted fractions were then pooled in three successive sections: Section 1 - fractions 17-50; section 2 - fractions 51-60; and section 3 - fractions 61-70.
  • fraction 51-60 was also tested for yeast activity and resulted in early activity development. Additionally, an aliquot of fraction 61-70 was tested for yeast activity and resulted in the strongest inhibitory effect among the three fractions. These data are not shown in the figures.
  • Fractions 19-32 showed a late activity development, as seen in Figure 10. That result indicates that fraction 19-32 may have a high molecular weight above 20,000d.
  • a matching protein in the brewer's barley extract elutes at the same position on GP chromatography. That protein is not abundant in the extract, and has a molecular weight above 12,000d because it does not release its activity on dialysis with 12,000 d cut-off tubings.
  • the yeast activity profiles of two leading proteins are shown comparatively in Figure 10.
  • Fractions 57-61 were tested for yeast activity and showed early activity development, as seen in Figures 8 and 9.
  • the position of this protein coincides with the position of brewer's barley HL-protein known to carry UV absorbing ligands at 265 nm.
  • Past tests have shown that the HL-protein releases its ligands in the presence of salt in a 0.5M concentration.
  • the HL-protein has a molecular weight of 14,000d.
  • Figure 9 shows comparative yeast fermentation activities of HL-protein and its matching component of GMM at Fractions 57-61.
  • the amount of HL-protein in the test was as low as 5 mg.
  • chromatographic fractionation of GMM retentate by gel permeation size exclusion revealed the presence of macromolecular species in the GMM preparation, most of which were proteins. Some of the proteins in the GMM retentate showed a significant inhibiting action on the yeast fermentation. Two macromolecular components were detected with apparent yeast fermentation activity. A peak in activity was located in the first portion of an elution pattern, which belonged to a molecular species passing through dialyzing membranes with a 12,000d cut-off.
  • L6 cells were grown as monolayers in Dulbecco's modified Eagle's medium (DMEM) containing ImM glutamine, ImM pyruvate and 25mM glucose. The medium was supplemented with 5% fetal calf serum (FCS). Experiments were undertaken in 48-well plates seeded from pre-confluent flasks with 50000 cells per well. The cells were grown to confluence and the medium was changed to DMEM containing 0.5% FCS and 2.5 mM glucose (L6 Myo Medium) for at least 76 hrs to induce differentiation of L6 cells and formation of L6 myotubes.
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • Myotubes were then incubated with test substances for 30 minutes - Protocol SMM, or in Dulbecco's PBS supplemented with 20mM Hepes and 2mM glucose without serum - Serum-free Medium (SFM) Protocol as described by Fryer et al. ((Activation of glucose transport by AMP-activated protein kinase via stimulation of Nitric oxide synthase, Diabetes, 49(12), 1978-85, 2000, Fryer LGD, et al).
  • SFM serum - Serum-free Medium
  • L6 cells treated with insulin in serum-free medium responded up to 40-50 % glucose uptake increase.
  • treatment of L6 with insulin in culture medium supplemented with 0.5% FCS (SSM) increased glucose uptake up to 100-120% by average.
  • Figure 12 shows the effect of permeate on glucose uptake in L6 cells.
  • Cells were cultured, and treated in L6 Myo Medium (SSM) - SSM Protocol or in serum-free medium (SFM - DPBS containing 20mM Hepes, 2mM glucose) - SFM Protocl as described by Fryer et al.
  • SSM L6 Myo Medium
  • SFM serum-free medium
  • Glucose uptake was measured by adding 30uM of 2-NBDG for 2' at 22C. Experiments were performed in duplicates.
  • Figure 13 shows the effect of AICA on the stimulation of glucose uptake and the effect of L-NMMA on the inhibition of glucose uptake. Similar effects are described by Fryer et al (Activation of glucose transport by AMP-activated protein kinase via stimulation of Nitric oxide synthase, Diabetes, 49(12), 1978-85, 2000, Fryer LGD, et al).
  • the permeate cells were pre-treated with L-NMMA for 30'in DPBS and subsequently treated with AICA for 30 minutes. Glucose uptake was measured after the treatment in 22C for 2'.
  • AMP-activated protein kinase AMP-activated protein kinase
  • AICAR Nitric Oxide Synthase
  • L-N-mono-methyl-L-arginine (1-NMMA) or L-N-nitro-L-arginine methyl ester (L-NAME) completely blocks the increase the glucose transport after activation of AMPK.
  • AICA was used because AICAR is not commercially available.
  • AICA is a precursor of AICAR and it is metabolized in muscle cells to AICAR resulting in increase glucose uptake.
  • L-NMMA has been used in the experiment to verify whether GMM-induced glucose transport in L6 cells is or is not inhibited by NOS inhibitors, thus mimicking AICAR - induced mechanism of increased glucose transport.
  • Fryer's experimental set up was adopted, consisting of pre-treatment of cells with inhibitor for 30 minutes in DPBS and after that with agonists like AICAR or in our case GMM for next 30 minutes at 37C before performing glucose uptake.

Landscapes

  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Diabetes (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Obesity (AREA)
  • Hematology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Child & Adolescent Psychology (AREA)
  • Botany (AREA)
  • Medical Informatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Emergency Medicine (AREA)
  • Endocrinology (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des compositions et des méthodes destinées à augmenter la fixation du glucose sur une cellule. Plus particulièrement, la présente invention se rapporte à des compositions contenant un composé extrait d'une plante présentant une activité comparable à l'AICAR ainsi qu'à des compositions contenant un composé non-insulinique qui présente une activité comparable à l'AICAR et active l'AMPK d'une cellule.
EP02750593A 2001-03-08 2002-03-08 Compositions et methodes pour une fixation non-insulinodependante du glucose Withdrawn EP1372725A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
WOPCT/US01/07527 2001-03-08
PCT/US2001/007527 WO2001066146A1 (fr) 2000-03-08 2001-03-08 Procedes et compositions de supplements alimentaires
PCT/US2002/007199 WO2002072148A1 (fr) 2001-03-08 2002-03-08 Compositions et methodes pour une fixation non-insulinodependante du glucose

Publications (2)

Publication Number Publication Date
EP1372725A1 true EP1372725A1 (fr) 2004-01-02
EP1372725A4 EP1372725A4 (fr) 2004-04-07

Family

ID=21742385

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02750593A Withdrawn EP1372725A4 (fr) 2001-03-08 2002-03-08 Compositions et methodes pour une fixation non-insulinodependante du glucose

Country Status (4)

Country Link
EP (1) EP1372725A4 (fr)
JP (1) JP2005501809A (fr)
CA (1) CA2439774A1 (fr)
WO (1) WO2002072148A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109295068B (zh) * 2018-09-14 2021-04-09 昆明理工大学 一种三七类甜蛋白基因PnTLP2及应用
JP2020115816A (ja) * 2019-01-25 2020-08-06 株式会社東洋新薬 糖取り込み促進用組成物

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5658889A (en) * 1989-01-24 1997-08-19 Gensia Pharmaceuticals, Inc. Method and compounds for aica riboside delivery and for lowering blood glucose
US5932258A (en) * 1998-04-06 1999-08-03 The Iams Company Composition and process for improving glucose metabolism in companion animals
WO2001066146A1 (fr) * 2000-03-08 2001-09-13 Vdf Futureceuticals Procedes et compositions de supplements alimentaires
US20010051195A1 (en) * 2000-03-08 2001-12-13 Miljkovic Dusan A. Methods and compositions for increasing fermentation of a microorganism

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6365176B1 (en) * 2000-08-08 2002-04-02 Functional Foods, Inc. Nutritional supplement for patients with type 2 diabetes mellitus for lipodystrophy

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5658889A (en) * 1989-01-24 1997-08-19 Gensia Pharmaceuticals, Inc. Method and compounds for aica riboside delivery and for lowering blood glucose
US5932258A (en) * 1998-04-06 1999-08-03 The Iams Company Composition and process for improving glucose metabolism in companion animals
WO2001066146A1 (fr) * 2000-03-08 2001-09-13 Vdf Futureceuticals Procedes et compositions de supplements alimentaires
US20010051195A1 (en) * 2000-03-08 2001-12-13 Miljkovic Dusan A. Methods and compositions for increasing fermentation of a microorganism

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Blood sugar depressant for diabetes treatment - contains water-soluble fraction from green leaf juice of barley and/or wheat" DERWENT, XP002229033 *
MAHDI G S ET AL: "ROLE OF CHROMIUM IN BARLEY IN MODULATING THE SYMPTOMS OF DIABETES" ANNALS OF NUTRITION AND METABOLISM, KARGER, CH, vol. 35, no. 2, 1991, pages 65-70, XP009004930 ISSN: 0373-0101 *
PICK M E ET AL: "BARLEY BREAD PRODUCTS IMPROVE GLYCEMIC CONTROL OF TYPE 2 SUBJECTS" INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION, CARFAX PUBLISHING LTD, GB, vol. 49, no. 1, January 1998 (1998-01), pages 71-78, XP009004933 ISSN: 0963-7486 *
See also references of WO02072148A1 *

Also Published As

Publication number Publication date
JP2005501809A (ja) 2005-01-20
EP1372725A4 (fr) 2004-04-07
CA2439774A1 (fr) 2002-09-19
WO2002072148A1 (fr) 2002-09-19

Similar Documents

Publication Publication Date Title
JP4353982B2 (ja) 糖尿病処置用相乗作用組成物
US9512453B2 (en) Method for preparing novel processed ginseng or an extract thereof, the usually minute ginsenoside content of which is increased
Han et al. Isolation and hypoglycemic effects of water extracts from mulberry leaves in Northeast China
JP3020387B2 (ja) 抗ウイルス物質
US7320806B2 (en) Compositions and methods for treating NIDDM and other conditions and disorders associated with AMPK regulation
US6964785B2 (en) Herbal composition for improving anticancer activity, immune response and hematopoiesis of the body, and protecting the body from oxidative damage, and the method of preparing the same
KR20080080127A (ko) 파세러스 불가리스 추출물, 그 용도 및 이를 함유하는배합물
JP2006347952A (ja) リパーゼ活性化作用を呈するアントラキノン誘導体、それからなる抗肥満薬、食品製剤及び化粧品
US20080166433A1 (en) Methods and Composition For Dietary Supplements
Sarkar et al. Evaluation of in vitro anti diabetic activity of two mangrove plant extracts: Heritiera fomes and Sonneratia apetala
CN110693025B (zh) 一种多功能成分协同降血糖的组合物及其应用
EP1372725A1 (fr) Compositions et methodes pour une fixation non-insulinodependante du glucose
US20040170705A1 (en) Compositions and methods for non-insulin glucose uptake
AU2001249128A1 (en) Methods and compositions for dietary supplements
AU2002306682A1 (en) Compositions and methods for non-insulin glucose uptake
KR101385191B1 (ko) 치커리 추출물의 근육 손상 예방, 치료 또는 개선을 위한 용도
US20190091287A1 (en) Plant fractions having anti-pathogenesis properties
KR20110053141A (ko) 발아현미 추출물을 포함하는 항당뇨 조성물
CN108250258B (zh) 一种利用发酵与超声波提取技术制备的新黄酮糖苷
JP2009520715A (ja) ファセオラス・ブルガリス抽出物、それらの使用、およびそれらを含む組成物
CN100548315C (zh) 家蚕保健品及其制备方法
Obochi et al. Efficacy and suitability of lectin from Mucuna sloanei seeds extracts as a cell receptor signal inducer
CN118078873A (zh) 苦苣菜提取物在制备降血压药物中的应用
Lae et al. A study on the biochemical properties of Triticum aestivum Linn.(wheatgrass)
CN116115731A (zh) 一种降血糖的组合物、制剂及其应用

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20031007

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

A4 Supplementary search report drawn up and despatched

Effective date: 20040219

RIC1 Information provided on ipc code assigned before grant

Ipc: 7A 61P 3/10 B

Ipc: 7A 61K 35/78 A

RIN1 Information on inventor provided before grant (corrected)

Inventor name: PIETRZKOWSKI, ZBIGNIEW,VDF FUTURECEUTICALS

Inventor name: HRANISAVLJEVIC, JOVAN,VDF FUTURECEUTICALS

Inventor name: MILJKOVIC, DUSAN

17Q First examination report despatched

Effective date: 20050512

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20051122