WO2007129951A1 - Sensibilite des virus aux nucleosomes vegetaux - Google Patents

Sensibilite des virus aux nucleosomes vegetaux Download PDF

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Publication number
WO2007129951A1
WO2007129951A1 PCT/SE2007/000421 SE2007000421W WO2007129951A1 WO 2007129951 A1 WO2007129951 A1 WO 2007129951A1 SE 2007000421 W SE2007000421 W SE 2007000421W WO 2007129951 A1 WO2007129951 A1 WO 2007129951A1
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Prior art keywords
plant
histones
chromatin
proteinaceous
proteinaceous component
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PCT/SE2007/000421
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English (en)
Inventor
Lars-Olof Hedén
Uif Rothman
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Svenska Miljöbolaget SVV AB
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Priority to EP07748086A priority Critical patent/EP2020858A4/fr
Priority to CA002651239A priority patent/CA2651239A1/fr
Publication of WO2007129951A1 publication Critical patent/WO2007129951A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/20—Fabaceae or Leguminosae [Pea or Legume family], e.g. pea, lentil, soybean, clover, acacia, honey locust, derris or millettia
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/40—Liliopsida [monocotyledons]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/40—Liliopsida [monocotyledons]
    • A01N65/44—Poaceae or Gramineae [Grass family], e.g. bamboo, lemon grass or citronella grass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3472—Compounds of undetermined constitution obtained from animals or plants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3526—Organic compounds containing nitrogen
    • 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)
    • A61K36/31—Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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)
    • A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals

Definitions

  • the present invention relates to the use of a proteinaceous component isolated from plant chromatin. More precisely, the invention relates to the use of a proteinaceous component isolated from plant chromatin, after dissociation of the same, as an antiviral agent, as well as a method of producing the same.
  • phospholipid transfer proteins which are able to transfer phospholipids between membranes.
  • Antimicrobial phospholipid transfer proteins have been reported from a range of plant species including cereals, and these proteins vary in their activity against different pathogens. For example, in US 5 698 200 it is shown that a plant part can be protected from a plant pathogenic bacterium by means of an aqueous extract obtained from malted cereal grain.
  • antimicrobial peptides are found in all species of life, ranging from plants and insects to animals, including molluscs, crustaceans, amphibians, birds, fish, mammals, including humans.
  • peptides interact directly with bacteria and kill them. They are termed antimicrobial because they have unusually broad spectra of activity including the ability to kill or neutralize Gram-negative and Gram-positive bacteria fungi (including yeast), parasites (including planaria and nematodes), cancer cells, and even enveloped viruses like HIV and herpes simplex virus. In general, these agents range in length from as few as 12 amino acids to molecules with over 70 residues. More than 500 such peptides have been discovered.
  • the mode of antimicrobial action of the almost always cationic antimicrobial peptides has been studied in detail among such peptides as melittin, magainin, gramicidin, cecropin, and defensins.
  • the antimicrobial molecules also generally damage the membranes of the organisms that they attack.
  • the cationic antimicrobial peptides have been found to possess bactericidal activity in vitro as well as in vivo. They kill very rapidly, do not easily select resistant mutants, are synergistic with conventional antibiotics, other peptides as well as lysozyme, and are able to kill bacteria in animal models.
  • antimicrobial peptides of animal origin are now developed as new antibiotic drug.
  • examples are the synthetic version of magainin (pexiganan) and the analogue of a protegrin, an antimicrobial peptide initially isolated from pig neutrophils.
  • ubiquicidin a murine macrophage protein, ubiquicidin appears to be the same as the ribosomal protein S30.
  • two of the antimicrobial peptides in the stomach of bullfrog ⁇ Rana catesbeina) are derived from the N-terminus of pepsinogen.
  • a biocide peptide, named buforin I has been isolated from stomach tissue of an Asian toad (BBRC 218:408, 1996).
  • the amino acid sequence of the 39 amino acid long peptide was found to be identical with 37 of the 39 amino- terminal residues of the Xenopus histone H2A.
  • the whole protein molecule can exhibit an antimicrobial potential. Antimicrobial activity has been detected in acid extracts of liver, intestine, and stomach of atlantic salmons (BBRC 284:549, 2001).
  • the corresponding antimicrobial protein can be isolated from salmon liver using acid extraction followed by ammonium sulfate precipitation, large-scale gel chromatography (gel filtration), reverse-phase HPLC, and size exclusion HPLC.
  • SAM salmon antimicrobial
  • the mammalian histone H1 protein from bovine thymus is used in antimicrobial compositions for treating microbial infections in different eukaryotic organisms.
  • proteins having other well-established functions appear to exhibit a second property by being antimicrobial.
  • bovine proteins especially proteins from bovine thymus
  • Bovine material should be avoided since such a material can be contaminated with deleterious virus, especially hepatitic viruses, or other pathogenic agents, for example priones.
  • Bovine material - whether contaminated or not - must be subjected to extremely strict tests when intended to be used in connection with humans.
  • the isolation of new alternative antibiotics involves the collection of specified animal organs or tissue, followed by complex purification procedures in order to obtain a product that can be used in connection with human beings or domestic animals.
  • WO 03/017769 relates to the use of a proteinaceous component isolated from plant chromatin, after dissociation of the same, as an antimicrobial agent, the proteinaceous component having an apparent molecular weight between 10 and 20 kD.
  • the proteinaceous plant component is produced by means of a method comprising the steps of homogenizing a plant material in order to expose its plant chromatin, dissociating the plant chromatin with a dissociating agent under hydrophobic conditions, and separating the dissociated plant chromatin into individual fractions, one comprising the proteinaceous plant component, by means of a hydrophobic interaction separation procedure.
  • the said WO 03/017769 relates to antimicrobial treatment, where the microbes are bacteria, or fungus (yeasts).
  • the present invention relates to an antiviral agent, and in particular to the use of a proteinaceous component isolated from any karyotic chromatins after dissociation of the same, as an antiviral agent, the very proteinaceous nucleosomal main component and its subfractions.
  • a proteinaceous component isolated from any karyotic chromatins after dissociation of the same as an antiviral agent, the very proteinaceous nucleosomal main component and its subfractions.
  • Another purpose of the invention is to provide an antiviral agent, whereby the risk is avoided of passing on infectious agents in the food chain or being pathogenic to man and/or animals and/or plants.
  • Still another purpose is to provide an antiviral agent that is tasteless when used in connection with food.
  • a further purpose of the invention is to provide a method of producing an antiviral agent, in which cheap starting materials are utilized.
  • Yet a further purpose is to provide a method of producing an antiviral agent in a practically unlimited scale.
  • Still yet a further purpose is to provide a method of producing an antiviral agent, which does not require investments manufacturing in plants for microbiological fermentation.
  • a method in which in a simple and rational way allows for the production of a proteinaceous component which can be used as an antiviral product, for example as a drug, a full preserving agent during manufacturing and transport, a functional food, such as a probiotic or prebiotic food, and/or neutraceutical additive as well as an animal feed additive.
  • a proteinaceous component which can be used as an antiviral product, for example as a drug, a full preserving agent during manufacturing and transport, a functional food, such as a probiotic or prebiotic food, and/or neutraceutical additive as well as an animal feed additive.
  • a proteinaceous component can be prepared with surprisingly ease from an initially inert starting material comprising plant chromatin.
  • DNA is separated from basic nuclear plant chromatin.
  • the plant chromatin is obtained from plant grains, embryos or seeds.
  • plant material is obtained from the commercial commodities oat, wheat, barley, rye, com, rye wheat, rice, rape, soy bean, millet, sorghum, milo, or buck wheat.
  • any chromatin containing plant material cauliflower, spinach, green grass
  • weeds edible vegetables, leftovers from the vegetable oil industry, gardening or forestry waste can be used for the preparation of an antiviral proteinaceous component on a large scale.
  • Another important source is the commercial yeasts. However, fresh plant material is seldom optimal for the isolation and recovery of plant chromatin.
  • the plant chromatin used for the isolation of the proteinaceous component should be a heterochromatin (silent chromatin or "junk" DNA.
  • the heterochromatin is hypoacet- ylated (deacetylated) chromatin, which assumes a more condensed structure than hyperacetylated chromatin due to a higher electropositive charge.
  • chromatin starting material for further specific protein extraction is also dependent on plant cell tissue location and state of differentiation. For example, a tissue comprised of small cells will have a higher cell density, and therefore is likely to contain more nucleic acids and accompanying antibacterial proteinaceous component than another same amount of tissue comprising larger cell size.
  • the amount of DNA per haploid cell as measured in the number of base pairs is referred to.
  • the variation in DNA content of an organism is reflected by its DNA c-value or basal genome size.
  • the c-value is defined as the content of DNA as measured by weight or number of nucleotides in a single copy of the entire sequence of DNA found within cells of that organism. It is the amount of nuclear DNA in its unreplicated haploid or gametic nucleus, irrespective of the ploidy level of the taxon.
  • the c-value equals the genome size in diploid species, but always exceeds genome size in polyploid species.
  • plant seedling root-tips are the superior starting material for chromatin extraction and subsequent downstream isolation of the proteinaceous component according to the invention. Such raw material is readily available in unlimited quantities, as being a waste product during the manufacturing of brewery malt and wheat germ oil.
  • plant raw materials of mitotically dividing cells under optimal growing conditions are also suitable for the preparation of a proteinaceous component according to the invention.
  • Any germinating sprouts and rootlets or germs in germination phase can be used.
  • grains of one of the four kinds of cereals are used, which are allowed to germinate.
  • a cost effective raw material to be used according to the present invention is what is called green malt, which is a starting material for beer production.
  • the brewery industry produces green malt from barley, which after moisturizing is allowed to germinate for six days (malting).
  • This industrially produced green malt, or by-products thereof (rootlings) can according to the invention be used for the production of an antiviral proteinaceous component.
  • rootlings of diploid corn and barley (DNA c-value of 5,000 Mbp) as well as onion (DNA c-value of 18,000 Mbp) are suitable starting extraction materials.
  • the antibacterial proteinaceous component is extracted from a chromatin source, the DNA c-value of which exceeds 3,000 Mbp.
  • the starting material of the purification procedure comprises plant chromatin isolated from proliferating plant cells in S-phase. Germinating seeds (grains) with their rootlings as well as young leaves thus contain a large number of cells in S-phase.
  • the method comprises the steps of
  • the plant material is first homogenized.
  • homogenization means a disruption of the plant material cell walls in such a way that the chromatin of the plant is exposed and a homogenate is obtained as a slurry.
  • the cell walls may be disrupted by any of a number of methods known to those skilled in the art including, but not limited to, high shear mixing, sonication, mechanical disruption, explosion by pressure etc.
  • the cell walls are disrupted by means of a suitable device, whereby a homogenate is obtained.
  • the plant chromatin in the homogenate is then dissociated by means of a dissociating agent in an aqueous solution thereof under hydrophobic conditions. Such conditions are those that promote hydrophobic interactions.
  • Suitable dissociating agents are urea, guanidinium chloride, and a chloride salt.
  • the chloride salt is sodium chloride of high ionic strength.
  • the histones are isolated as nucleosomes in a solution having a chloride concentration of above 0.3 M, preferably 0.5 M or higher. This nucleosome mixture may be inert with regard to microbiological effect, but will become activated by diluting the solution to a chloride concentration of 0.3 M or below. Thereby free histones will dissociate and go into solution.
  • the purification procedure of green malt is commenced by the homogenisation of the malt in an almost saturated salt solution comprising 4 M sodium chloride.
  • the high ionic strength dissociates the chromatin as well as nucleosomes, a simultaneous degradation of proteinaceous material by proteases at the same time being prevented.
  • the homogenisation is performed in the presence of a hydrophobic matrix.
  • the homogenate can then be sieved on a sieve or a wire net or the like in order to remove cell debris or other particles from the plant chromatin, which are retained thereon. In this way a solution is obtained that facilitates a subsequent purification of the dissociated plant chromatin.
  • the dissociated plant chromatin is then separated into individual fractions, one comprising the proteinaceous plant component having antiviral activity.
  • the separation is preferably performed by means of a hydrophobic interaction separation procedure.
  • the hydrophobic interaction separation procedure is hydrophobic chromatography.
  • the supernatants are combined and passed through four consecutive nylon fabrics with defined mesh (2 mm, 1 mm, 0.5 mm, and finally 0.3 mm).
  • the volume is adjusted to 1 ,000 ml and 3 M ammonium sulphate is added dropwise to a final concentration of 50 mM with stirring to precipitate the chromatin.
  • the pellet is homogenized in 200 ml TBM and centrifuged as above. The homogenization and centrifugation is repeated four times. Finally, the precipitated material is homogenized in 200 ml 0.1xSSC+5 mM NaHSO 3 .
  • the histones in the chromatin preparation are extracted by dropwise addition of 0.5 M H 2 SO 4 with stirring to a final concentration of 0.1 M. Leave at +4 0 C with stirring for 4 hours. Centrifuge at 10,000xg, +4 0 C for 10 minutes and recover the supernatant. The pellet is reextracted with 50 ml 0.1 M H 2 SO 4 with stirring at+4°C for 4 hours and centrifuged as above. The supernatants are combined and concentrated with polyethylene glycol 6,000 and dialyzed against 0.1% acetic acid using dialysis tubing with a molecular cut off of 3,000 Dalton. The dialyzed material is stored at -2O 0 C
  • Suitable separation procedures are partition in polymeric systems, such as partition chromatography, counter current distribution, and gas aphron partition.
  • the separation of the dissociated chromatin components can alternatively be performed on columns with metal chelate gels or immobilized heparin.
  • the functional ligand of the matrix used for the hydrophobic interaction and/or separation procedure should be an ether, an isopropyl, a butyl, or an octyl group.
  • a phenyl group should be avoided.
  • the functional ligand is a butyl group on an agarose matrix which is cross linked to 4%.
  • a ligand density of 40-50 ⁇ mol/ml is the achieved, which results in a binding capacity of 7 mg IgG per ml.
  • a hydrophobic matrix is added batch wise to the solution obtained, the hydrophobic matrix being an hydrophobic interaction chromatography gel (HIC) containing active butyl groups.
  • HIC hydrophobic interaction chromatography gel
  • Suitable matrixes are Novarose® S-Butyl 1000/40 from Inovata AB, Bromma, Sweden, and Butyl Sepharose® 4 from Amersham Pharmacia Biotech, Sweden.
  • the hydrophobic matrix is then washed with the high ionic strength salt solution, DNA being washed out. Then the matrix is poured into a column and subjected to a stepwise gradient elution with decreasing ionic strength of sodium chloride.
  • a distinct antiviral proteinaceous component is eluted at a concentration of 1 M NaCI.
  • the proteinaceous component can be further purified by means of a conventional method suitable for purification of peptides/proteins.
  • a conventional method suitable for purification of peptides/proteins include centrifugation, precipitation at the isoelectric point, phase separations, ultrafiltration, gel chromatography (size exclusion chromatography), ion exchange chromatography or HPLC, as well as a combination of such methods.
  • the subsequent separation procedure is gel chromatography or ion exchange chromatography.
  • a preparative gel chromatography step is accomplished in a column packed with a gel having an exclusion limit of 100 kD.
  • the column is equilibrated with distilled water before being loaded with the fraction of 1 M NaCI exhibiting antibiotic activity.
  • the column is then eluted with distilled water or ammonium acetate.
  • a desalting and purification is obtained at the same time in one and the same step.
  • the proteinaceous component can be concentrated to dryness, for example by means of lyophilization, without any further purification steps.
  • a protein fraction having an apparent molecular weight between 10 and 20 kD was isolated, which exhibited antiviral properties.
  • a complexing agent such as heparin, alginic acid, phytic acid, polyphosphate or a vanadinium compound, can also be used as a dissociating agent, provided that it dissociates the plant chromatin into its individual components.
  • Alginic acid is especially preferred as a dissociating agent, alginate complexes with the viricidal active proteinaceous component being formed. Such complexes can be used with the aim of purification or be used as such for slow release of antiviral activity therefrom.
  • other antiviral active proteinaceous components may be obtained from other plant materials by means of other purification procedures after the elution from the hydrophobic matrix. This is due to the fact that proteins from different biological materials exhibit different post-synthetical modification patterns that reflect cellular activities of the plant material.
  • a separation pattern should be influenced by the degree of for example acetylation, phosphorylation, methylation, ubiquitination, glycosylation, as well as ADP-ribosylation of a proteinaceous component obtained according to the invention.
  • the isolated proteinaceous component from plant chromatin can subsequently be chemically modified.
  • modifications include changes in molecular weight and/or acetylating level and would result in preparation forms having a more specific biological activity.
  • the simple inventive purification method allows for the production of an antiviral proteinaceous component in a practically unlimited scale.
  • the process yield is more than 1 g protein from 1 kg of raw material (for example rootlings).
  • germinating seeds with maximum protein synthesis the yield can be maximized, as is shown for example by malting for six days.
  • the natural chromatin protein synthesis is maximal and can represent up to 80% of the total protein synthesized.
  • the proteinaceous component isolated from plant chromatin according to the invention as an antiviral agent can be intensified together with one or more antivirally synergistic agents.
  • the proteinaceous component When the proteinaceous component is to be used as an antiviral agent, it can be formulated in buffered aqueous media containing a variety of salts and buffers.
  • the salts are alkali and alkaline earth halides, e.g. sodium chloride, potassium chloride, or sodium sulphate.
  • Various, such as buffers may be used, such as citrate, phosphate, HEPES, Tris or the like to the extent that such buffers are physiologically acceptable for its purpose.
  • excipients or other additives may be used, when the proteinaceous component is formulated as a lyophilized powder, for subsequent use in solution.
  • the excipients may include various polyols, inert powders or other extenders.
  • the inventive use also includes a composition that comprises the purified proteinaceous component in a biocidal concentration and an amount effective to kill bacteria or fungi and a suitable carrier.
  • a composition that comprises the purified proteinaceous component in a biocidal concentration and an amount effective to kill bacteria or fungi and a suitable carrier.
  • Such compositions may be used in numerous ways to combat viruses, for example in household or laboratory antiviral formulations using carriers well-known in the art.
  • compositions will have different degrees of activities towards different viruses.
  • Effective amounts to be used for killing harmful viruses may be readily determined by those skilled in the art.
  • the proteinaceous component according to the present invention may also be combined with other proteins to act as preservatives in order to protect the proteinaceous component against proteolytic degradation.
  • inventive proteinaceous component or compositions may be used as preservatives or disinfectants in a wide variety of formulations, such as contact lens solutions, ointments, shampoos, medicaments, foods, and the like.
  • the amount of proteinaceous component may vary depending upon the nature of other components, the degree of antiviral protection required, and the intended use of the composition.
  • the proteinaceous component can for example be used together with a suitable carrier in a composition for disinfection and cold sterilization of surfaces and as an adjuvant in food high-pressure pasteurization as well as in a composition as a water conservation agent, e.g. in pisciculture or to protect fishes in aquaculture.
  • the proteinaceous component can also be used in an amount effective to kill viruses when enclosed in packaging materials to be slowly released therefrom.
  • Histones prepared by acid extraction of wheat germ have been shown in laboratory to have antimicrobial activity against a large number of bacterial species, gram-positive as well as gram-negative strains.
  • the wheat germ histone preparations were found to inactivate the non-enveloped bacteriophages ⁇ X174, ⁇ , and MS2.
  • the results also suggest that the wheat germ histones are able to inactivate the fish pathogenic virus IPNV (infectious pancreatic necrosis virus).
  • Bacteriophage ⁇ X174 single stranded circular DNA genome
  • Bacteriophage MS2 single stranded linear RNA genome
  • Bacteriophage concentrations were determined as plaque forming units (pfu) on nutrient agar (for ⁇ X174) or tryptose agar (for ⁇ and MS2) using a top agar with 0.6% agar in tryptose broth. The strain used for the propagation of the phage was also used as indicator bacteria in the top agar.
  • the different phages were diluted to the appropriate concentration in phosphate buffered saline (PBS for ⁇ X174 and MS2, ⁇ -buffer for phage ⁇ ) or distilled water.
  • PBS phosphate buffered saline
  • the acid extracted histones were used either in 0.1 M acetic acid or dialyzed against several changes of PBS and diluted in PBS or in distilled water.
  • Bacteriophage and wheat germ histones were mixed on ice in plastic tubes and incubated in a water bath set to the appropriate temperature and for the time indicated below. After the incubation, the number of pfu was determined as described above.
  • the agar plates were incubated over night at 3O 0 C for ⁇ X174 and at 37 0 C for ⁇ and MS2.
  • the salmon fish cell line Xxx was grown in micro titre plates with YYY minimal medium supplemented with 5% heat inactivated fetal calf serum. The cells were grown at 2O 0 C in a CO 2 incubator (10% CO 2 ). The virus IPNV and the wheat germ histones were diluted in PBS. Virus and histones were mixed in micro titre plate wells and incubated at 37° C for two hours after which 20 ⁇ l was added to micro titre plate wells with fish cells and 200 ⁇ l growth medium and incubated as for propagation of the fish cells. Viral infection of the cells was determined as cytophatogenic effect using a microscope. The cells were observed daily for three days and finally fixed as described by Carlsson et al. (3).
  • Bacteriophage ⁇ was diluted to 2x10 4 pfu/ml. Equal volumes of the diluted phage and acid extracted histone (4,530 ⁇ g/ml) in 0.1 IVl acetic acid was mixed and incubated at room temperature. At different time points, pfu/ml was determined. As a control, the same volume of diluted phage was mixed with an equal volume of 0.1 M acetic acid.
  • the histone preparation was diluted in 0.1 M acetic acid and equal amounts of diluted histones and phage ⁇ (2x10 4 pfu/ml) were mixed on ice and incubated at room temperature for 60 minutes. Pfu was determined as above and the results are presented in Fig. 2.
  • the temperature dependence of the phage inhibiting properties of the histones was analyzed by mixing phage ⁇ X174 diluted to ⁇ 4x10 6 pfu/ml in distilled water and different concentrations of wheat germ histone diluted in water. Incubations were done for 60 minutes at different temperatures before determination of pfu.
  • Table 2 show that at 10 0 C sixteen times more histones is needed to achieve the same degree of phage reduction compared to at 3O 0 C in 60 minutes. However, a 5, 000-fold reduction of pfu was obtained at a concentration of 22.7 ⁇ g/ml of histones when incubated at 1O 0 C for 60 minutes.
  • the antiviral activity of wheat germ histones was tested on the small icosahedral RNA phage MS2. Since MS2 is a male specific phage, i.e. uses sex pili as the receptor, the F + strain E. coli C66 was used for the propagation of the virus as well as for the determination of pfu. Phage MS2 was diluted to 4x10 6 pfu/ml in PBS. Diluted phage (0.4 ml) was mixed on ice with 0.1 ml wheat germ histpne at different concentrations.
  • the difference in rate of phage ⁇ inhibition in comparison to phage ⁇ X174 can be ascribed to the different temperatures used.
  • the assay of ⁇ X174 as described in Fig. 3 was performed at 37 0 C while the ⁇ inhibition experiment was performed at room temperature.
  • the temperature dependency for inactivation is further illustrated by the data in Table 2 below.
  • plaque morphology as shown in Fig. 5 was unexpected.
  • Phage ⁇ X174 normally forms large plaque due to the small size of the phage particle.
  • the plaque morphology suggests that the virus infection in the small sized plaques is due to a delay of the infection. This can be explained by a mechanism where the histones bound to the phage particles are "delivered" to the cells where the histones slow down the metabolic activity of the bacterial cells. This inhibition of metabolism is overcome with time provided that a sufficient low amount of histones have been introduced into the cells.
  • the whole mixture of plant histones works as an antiviral agent, and not only the part components, i.e., the core histones and the H1 histone.
  • the core histones and the H1 histone At the preparation of the histones these are soluble during acid extraction but natural nucleosomes are reconstituted while the ion strength declines during the final dialysing phase. This means that the product will obtain a stable form which can be stored and transported.
  • the antiviral effect might be due to a structural change on virus capsidal level changing the docketing ability of the virus.
  • the molecular weight of the H1 is between 40 and 45 kDa when analysing the molecular weight using SDS-PAGE. As the histones are negatively charged an acidic urea gel electrophoresis may provide a lower molecular weight.
  • Phage ⁇ X174 diluted in distilled water to about 3x10 e pfu/ml was mixed with wheat germ histones (dialyzed against PBS) diluted in distilled water and incubated at different temperatures for 60 minutes. Plaque forming units in the different mixtures were done as described in Material and Methods section above.
  • the above reported antiviral activity of acid extracted wheat germ histones has further been extended to include the single-stranded DNA bacteriophage M 13 and the mammalian Adenovirus Ad2.
  • the toxicity of the histones on mammalian HeLa cells has been analyzed by measuring the metabolic activity of cells exposed to histones.
  • the antiviral and antibacterial activities of heat-treated histones were analyzed.
  • Bacteriophage M13mp18 (Yanisch-Perron et al., 1985) is a filamentous single-stranded circular DNA virus using F-pili as the receptor in the infection of bacteria.
  • the phage was propagated using Escherichia coli JM103 (Yanisch-Perron et al., 1985) as the host strain.
  • Phage M13mp18 was diluted in10 mM sodium phosphate buffer, pH 7.0 (NAPB) to about 3x10 5 plaque forming units/ml (pfu/ml). From this dilution, 450 ⁇ l was mixed with 50 ⁇ l wheat germ histones diluted in NAPB. After 60 minutes incubation at 37 0 C the mixture was chilled on ice and plaque forming units was determined as described before (Yanisch-Perron et al., 1985) with exponentially growing E. coli JM103 as the indicator strain. As a control, 50 ⁇ l NAPB replaced wheat germ histones.
  • Costar (Corning Inc.) 48 wells microtiter plates were seeded with about 2x10 4 HeLa S3 cells in Eagle's minimal essential medium with 5% fetal bovine serum and incubated at 37 0 C in 5% CO 2 to about 80% confluency.
  • Adenovirus type 2 (Ad2) (1.7x10 12 virions/ml, about 3% infectious) is diluted in NAPB to a multiplicity of infection of 4 (see below).
  • conjugated goat anti-rabbit antibodies (secondary antibody).
  • p-Nitrophenyl phosphate /1 mg/ml was used as the substrate for the alkaline phosphatase. Absorbance at 405 nm was recorded after two hours at 37 0 C.
  • Wheat germ histones diluted in NAPB is added to HeLa S3 cells, in growth medium, in microtiter plate wells.
  • One set of cells were left with the histones for the entire 48 hours incubation and in one set of cells, the histone containg medium was replaced with histone free medium after one hour at 37 0 C and the incubated at 37 0 C for 48 hours.
  • 40 ⁇ l substrate EZ4U, non-radioactive cell proliferation & cytotoxicity assay, Biomedica, Germany
  • the absorbance at 450 nm was recorded after 60 minutes at 37 0 C.
  • the method is based on that living cells can reduce the uncoloured tetrazodium salt into the intensely coloured formazan. This reduction process requires functional mitochondria, which are inactivated within minutes after cell death.
  • Histones at a concentration of about 8758 ⁇ g/ml in 0.1% acetic acid was incubated at 100 0 C for 60 minutes and chilled on ice.
  • the antiviral activity after the heat treatment was analyzed by mixing 450 ⁇ l phage ⁇ X174 and 50 ⁇ l heat-treated or untreated histones diluted in NAPB. After 60 minutes at 37 0 C, the number of plaque forming units was determined with E. coli MM294 as indicator bacterium.
  • 50 ⁇ l 0.1% acetic acid was diluted in NAPB as for the histones and used as with the histones.
  • the antibacterial activity of wheat germ histones incubated at 100 0 C for different times was analyzed by radial diffusion assay as described before (Lehrer et al., 1991) with E. coli MM294 as the test organism..
  • Figure 9 the relative amount of formazan formed by HeLa cells in the presence of wheat germ histones is shown. The results show a marginal effect on the metabolism in the presence for one hour of up to 350 ⁇ g histones/ml. If the cells are exposed to the histones for 48 hours, a 50% reduction in formazan formation is shown. If this reflects a reduction of the metabolism in all cells or that a proportion of the cells are non-viable is not known presently. A staining for viable cells should be done to determine which possibility is true.
  • the antibacterial activity against E. coli MM294 of histones treated for different times at 100 0 C is shown in Figure 10.
  • the antibacterial activity remained the same even after 60 minutes at 100 0 C.
  • the results in Figure 11 show that also the antiviral activity was unaffected by incubation at 100 0 C for 60 minutes.
  • a reduction of the number of plaque forming units by four orders of magnitude was obtained for both the untreated and heat-treated histones.
  • the low concentration of acetic acid in the samples had no effect on the ability of the phage to infect the bacterial cells as shown in the control.
  • Figure 1 Time course inhibition of bacteriophage ⁇ multiplication. Equal volumes of phage ⁇ diluted in buffer to about 2x10 4 pfu/ml and acid extracted wheat germ histones were mixed and incubated at room temperature. At the time intervals indicated, 0.1 ml was taken for determination of pfu as described in Material and Methods section.
  • FIG. 4 Kinetics of phage ⁇ X174 inactivation by wheat germ histones.
  • FIG. 1 Size heterogeneity of phage ⁇ X174 plaques. The picture shows the plaque size heterogeneity in samples exposed to wheat germ histones.
  • Figure 7 Inhibition of M13 infection by wheat germ histones. Phage M13mp18 diluted as described in Material and Methods was incubated with different concentrations of wheat germ histones. After 60 minutes at 37 0 C the number of plaque forming units was determined with exponentially growing E. coli JM103 as the indicator organism. As a control, the same number of phage was incubated with NAPB.
  • HeLa S3 cells were exposed to increasing amounts of wheat germ histones for variable times. For one set of cells, the histone containing medium was replaced by histone free medium after one hour of incubation at 37 0 C. One set of cells was incubated at 37 0 C with histones for the entire 48 hour period. Reduction of tetrasodium salt was determined spectrophotometrically at 450 nm and expressed as the amount of formazan formed relative to control cells.
  • FIG. 10 Heat-resistance of wheat germ histones antibacterial activity. Aliquots of wheat germ histones were incubated at 100 0 C for different times as indicated and chilled on ice. The antibacterial activity was analyzed by radial diffusion assay with E. coli MM294 and the activity relative to non-heated sample is shown.

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Abstract

La présente invention concerne l'utilisation en tant qu'agent antiviral d'un composant protéique isolé de la chromatine végétale après la dissociation de cette dernière. Le procédé de production du composant protéique végétal comprend les étapes d'homogénéisation d'une matière végétale afin de libérer sa chromatine végétale, de dissociation de la chromatine végétale par un agent de dissociation dans des conditions hydrophobes, et de séparation de la chromatine végétale dissociée en fractions individuelles, dont l'une comprend le composant protéique végétal, au moyen d'une procédure de séparation par interaction hydrophobe. L'invention concerne en outre un procédé de traitement des infections virales dans des applications aussi bien techniques que pharmaceutiques.
PCT/SE2007/000421 2006-05-05 2007-05-03 Sensibilite des virus aux nucleosomes vegetaux WO2007129951A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001010901A2 (fr) * 1999-08-11 2001-02-15 Symbiotec Gmbh Compositions antimicrobiennes comprenant l'histone h1, trousses, et modes d'emploi desdites compositions
WO2003017769A1 (fr) * 2001-08-29 2003-03-06 Svenska Miljöbolaget SVV AB Agent antimicrobien

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001010901A2 (fr) * 1999-08-11 2001-02-15 Symbiotec Gmbh Compositions antimicrobiennes comprenant l'histone h1, trousses, et modes d'emploi desdites compositions
WO2003017769A1 (fr) * 2001-08-29 2003-03-06 Svenska Miljöbolaget SVV AB Agent antimicrobien

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE MEDLINE [online] LADYGINA M.E. ET AL.: "Comparative study of the antiviral properties of histones of animal and plant origin", XP008099851, Database accession no. (NLM34928) *
DATABASE MEDLINE [online] NAMBA T. ET AL.: "Development of antiviral therapeutic agents from traditional medicines", XP008099850, Database accession no. (NLM9778999) *
See also references of EP2020858A4 *
VOPROSY VIRUSOLOGII, November 1978 (1978-11-01) - December 1978 (1978-12-01), pages 686 - 690 *
YAKUGAKU ZASSHI: JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN, vol. 118, no. 9, September 1998 (1998-09-01), pages 383 - 400 *

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