WO2016072710A1 - 세리포리아 락세라타에 의해 생산되는 세포외다당체를 유효성분으로 함유하는 신경안정용 조성물 - Google Patents

세리포리아 락세라타에 의해 생산되는 세포외다당체를 유효성분으로 함유하는 신경안정용 조성물 Download PDF

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WO2016072710A1
WO2016072710A1 PCT/KR2015/011718 KR2015011718W WO2016072710A1 WO 2016072710 A1 WO2016072710 A1 WO 2016072710A1 KR 2015011718 W KR2015011718 W KR 2015011718W WO 2016072710 A1 WO2016072710 A1 WO 2016072710A1
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culture medium
laccerata
extracellular polysaccharide
weight
composition
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PCT/KR2015/011718
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English (en)
French (fr)
Korean (ko)
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박용만
신은지
김윤수
윤성균
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(주)퓨젠바이오농업회사법인
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Priority to CN201580059839.5A priority Critical patent/CN106998777B/zh
Publication of WO2016072710A1 publication Critical patent/WO2016072710A1/ko

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    • 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
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • 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
    • A61K36/06—Fungi, e.g. yeasts
    • 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
    • A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
    • 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
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/853—Lactobacillus

Definitions

  • the present invention relates to an extracellular polysaccharide produced by Ceriporia lacerata ( Ceriporia lacerata ) or a mycelial culture solution of Ceriporia laccerata comprising the same, a dry powder or extract thereof as an active ingredient .
  • the main organs involved are the cerebral limbic system (especially the hippocampus and the subject society), the cerebral cortex (frontal lobe, temporal lobe), hypothalamus, ascending reticular body and pituitary gland.
  • cerebral cortex frontal lobe, temporal lobe
  • hypothalamus ascending reticular body and pituitary gland.
  • Recent brain imaging studies have shown that anxiety is associated with disorders in the right hemisphere and other frontal, temporal, and occipital lobe disorders (Robert F. Scmidt, Human physiology , pp. 366; -40).
  • the study of therapeutic drugs for anxiety has attracted interest in the development of new drugs, especially glutamate receptor inhibitors, as well as the reevaluation of old drugs and the expansion of indications.
  • An ideal neurostabilizer is one that does not cause excessive drowsiness during the daytime, does not cause physical and mental dependence, and calms the patient.
  • Representative neurostabilizers currently available are benzodiazepine, diazepam, oxazepam, prazepam, lorazepam, alprazolam, helazepam, clona Such as clonazepam, and these drugs are also used for the purpose of sedation and sleep (Yoon Do Joon, Side Effects of Psychiatric Drugs, Journal of the Korean Medical Association, 38 (10), pp.1196-1202, 1995).
  • Benzodiazepines are the most commonly used neurostabilizers and are known to increase the affinity of GABA receptors, which are representative inhibitory neurotransmitters in the central nervous system, thus opening adjacent Cl ⁇ channels more often to increase the permeability of Cl ⁇ ions.
  • This benzodiazepine is effective immediately, but due to habits and addictions, symptoms may recur or withdraw if the drug is not used according to a specialist's treatment.
  • Other side effects include drowsiness, ataxia, orthostatic hypotension, respiratory depression, headache, and chronic sleep. Disorders, liver disease and the like have been reported (Mary J. Mycek et al. , Pharmacology 2nd edition, Lipincott Williams & Wilkins, pp. 89-93, 2000). Therefore, the research to develop a neurostable material with no side effects has been actively conducted.
  • Ceriporia laccerata is a type of white fungus and is known to perform cometabolism called lignin decomposition in order to use carbon sources such as cellulose, hemicellulose, other polysaccharides, and glycerol in an ecosystem.
  • lignin decomposition cometabolism
  • carbon sources such as cellulose, hemicellulose, other polysaccharides, and glycerol
  • Seriforia Lakseratta was first reported to academics in 2002, the study of its industrialization is still incomplete.
  • the present inventors have found that the mycelium culture medium, the dry powder or extract thereof of the extracellular polysaccharide produced by Seriphoria laccerata or the same, including the same, have a neurostable effect, which is used as an active ingredient.
  • the present invention relating to a neurostable composition is completed.
  • An object of the present invention is to provide a neurostable composition containing a pharmacologically active ingredient produced by Ceriporia laccerata.
  • Still another object of the present invention is to provide a health functional food having a neurostable effect containing a pharmacologically active ingredient produced by Ceriporia laccerata.
  • the present invention is an extracellular polysaccharide produced by Seriporia laccerata; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or it provides a neurostable composition containing the extract of the mycelia culture medium as an active ingredient.
  • the present invention is an extracellular polysaccharide produced by Seriporia laccerata; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or it provides a health functional food having neurostable efficacy containing the extract of the mycelia culture medium as an active ingredient.
  • the present invention is an extracellular polysaccharide produced by Seriporia laccerata; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or it provides a neurostable method comprising administering the extract of the mycelium culture to a subject in need of neurostable.
  • the present invention is an extracellular polysaccharide produced by Ceriporia laccerata for use in the manufacture of a medicament for neurostable; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or it provides a use of the extract of the mycelium culture.
  • 1 is a graph showing the change rate (% inhibition) of intracellular calcium concentration when the EPS concentration is 100 ug / mL.
  • the present invention is an extracellular polysaccharide produced by Ceriporia lacerata ( Ceriporia lacerata ); Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or it provides a neurostable composition containing the extract of the mycelia culture medium as an active ingredient.
  • the extracellular polysaccharide is about 40 to 60% by weight sugar and about 30 to 40% by weight protein, about 40 to 50% by weight sugar and about 32 to 38% by weight protein, or It may comprise about 43 to 47% by weight of sugar and about 33 to 36% by weight of protein, preferably about 45% by weight of sugar and about 34% by weight of protein.
  • the sugar may contain mannose, galactose and glucose.
  • the extracellular polysaccharide may have a molecular weight of about 100 to 150 kDa, about 110 to 140 kDa or about 115 to 125 kDa, and preferably may have a molecular weight of about 120 kDa.
  • the extracellular polysaccharide in one preferred embodiment, the extracellular polysaccharide,
  • the medium for the liquid culture of the seriporia laccerata mycelium in the step (a) is sugar, glucose, starch, hydrate, soybean meal, soybean meal, magnesium sulfate (MgSO 4 ), potassium phosphate (KH 2 PO 4 ), potassium diphosphate (K 2 HPO 4 ) and water, the hydrogen ion concentration (pH) may be 4.5 to 6.0.
  • the medium 0.2-3% by weight sugar, 0.2-3% by weight glucose, 0.2-4% by weight starch, 0.1-0.5% by weight moisture, 0.1-0.5% by weight wheat flour, soybean meal 0.2 to 3% by weight, 0.05 to 0.1% by weight of magnesium sulfate (MgSO 4 ), 0.05 to 0.25% by weight of potassium monophosphate (KH 2 PO 4 ), 0.05 to 0.25% by weight of potassium diphosphate (K 2 HPO 4 ) May comprise water.
  • MgSO 4 magnesium sulfate
  • KH 2 PO 4 potassium monophosphate
  • K 2 HPO 4 potassium diphosphate
  • the liquid culture in step (a) may be performed under a blue LED light source, and may be performed by maintaining a concentration of carbon dioxide at 1,000 to 2,000 ppm.
  • the liquid culture for example, at 20 ⁇ 25 °C, hydrogen ion concentration (pH) 4.5 ⁇ 6.0, light source blue LED, illuminance maintains 0.5 LUX and air is injected into 0.5 ⁇ 1.5 kgf / cm 2 and carbon dioxide It can be carried out for 8 to 13 days while maintaining the concentration of 1,000 to 2,000 ppm, it is preferable to be carried out for 10 days at 22 °C, pH 5.0, 1.0 kgf / cm 2 , 1,500 ppm conditions because the content of the extracellular polysaccharide is high. .
  • one excellent strain stored at 4 ° C. in a PDA (Potato dextrose agar) medium state was used at 25 ° C. in a shaker incubator using PDB (Potato dextrose broth) medium in an Erlenmeyer flask. It can be used to maintain a constant temperature and after 7 to 9 days incubation process.
  • the amount of mycelia to be added to the inoculum is preferably about 0.5% (w / v) based on the amount of the solution to be cultured.
  • the medium composition is a selective culture condition that forms the highest content of extracellular polysaccharides rather than the best nutritional ratio and environmental conditions for growth of the mycelia. It is preferable to apply.
  • the culture solution can be separated and purified into a mycelium and an aqueous solution.
  • the solution from which the mycelium is removed by centrifugation may be repeatedly purified using a multi-sheet filter press and a vibration centrifugal separator (PALLSEP), and then irradiated with ultraviolet (UV) light for 1 minute.
  • PALLSEP vibration centrifugal separator
  • UV ultraviolet
  • the solution needs to be kept sealed after removing oxygen, because if the mycelium is present in the solution, the mycelium grows by oxygen, which brings about a change in the content of the active ingredient.
  • the mycelia culture solution prepared in step (a) may be powdered by vacuum drying or lyophilization.
  • the drying is preferably carried out for 48 to 96 hours at a temperature of 40 °C or less, preferably 30 °C or less in order to prevent the disappearance of the active material.
  • drying in step (b) is preferable to use a vacuum freeze dryer rather than a vacuum dryer to set the evaporation temperature relatively high because the change in the effective material content is minimized.
  • step (c) after extracting the dry powder of the mycelium culture medium obtained in step (b) with a solvent to separate and prepare an extracellular polysaccharide which is an active ingredient of the composition according to the present invention. Specifically, 100 g of dry powder was added and 100 mL of distilled water was suspended, followed by centrifugation (8,000 rpm, 20 min) to add an extraction solvent corresponding to 2 to 3 times the amount of the supernatant thereof and a refrigerator (4 ° C). ) Can be left for 12 hours. After centrifugation (8,000 rpm, 20 min) again only the supernatant from the stationary material, the precipitate may be recovered to prepare crude extracellular polysaccharide. It is preferable to freeze-dry the crude extracellular polysaccharide at 30 ° C or lower.
  • the extraction solvent may be a solvent selected from the group consisting of water, ethanol, methanol, acetone, butanol and ethyl acetate or a mixed solvent thereof, preferably water or 50% (w / w) to 80% (w) / w) aqueous ethanol solution.
  • Extracellular polysaccharide produced by the seriporia laccerata according to the present invention Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium;
  • the neurostable composition containing the extract of the mycelium culture medium as an active ingredient may further include appropriate carriers, excipients, and diluents commonly used.
  • the extracellular polysaccharide may be included in 0.1 to 80% by weight, preferably 0.1 to 50% by weight based on the total weight of the composition, the mycelium culture medium, dry powder or extract thereof of the Seriphoria laccerata content of the extracellular polysaccharide It may be appropriately included in the amount corresponding to. However, the effective amount of the most preferred extracellular polysaccharide or the culture solution containing the same, its dry powder or extract can be appropriately adjusted according to the method of use and purpose of the composition.
  • compositions according to the invention can each be formulated and used according to conventional methods. Suitable formulations include, but are not limited to, tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, suppositories, and the like.
  • composition according to the invention can be prepared in a suitable formulation using a pharmaceutically inert organic or inorganic carrier. That is, lactose, sucrose, starch or derivatives thereof, talc, calcium carbonate, gelatin, stearic acid or salts thereof may be used when the formulation is a tablet, coated tablet, dragee and hard capsule.
  • a pharmaceutically inert organic or inorganic carrier that is, lactose, sucrose, starch or derivatives thereof, talc, calcium carbonate, gelatin, stearic acid or salts thereof may be used when the formulation is a tablet, coated tablet, dragee and hard capsule.
  • polyols of vegetable oils, waxes, fats, semisolids and liquids can be used.
  • water, polyols, glycerol, vegetable oils and the like can be used when the formulation is in the form of a solution or syrup.
  • composition according to the present invention may further include a preservative, a stabilizer, a wetting agent, an emulsifier, a solubilizer, a sweetener, a colorant, an osmotic agent, an antioxidant, and the like in addition to the above carrier.
  • the method of administering the composition according to the present invention can be easily selected according to the dosage form, and can be administered orally or parenterally.
  • the dosage may vary depending on the age, sex, weight, severity, and route of administration of the patient, but is generally in an amount of 5 to 500 mg / kg, preferably 100 to 250 mg / kg, based on the extracellular polysaccharide as an active ingredient.
  • the amount of may be administered once to three times a day.
  • the dosage does not limit the scope of the invention in any aspect.
  • composition according to the present invention not only provides an excellent neurostable effect, but also has little toxicity and side effects due to the drug, so that it can be used with confidence even for long term administration for neurostable purposes.
  • the compositions of the present invention can be used for the prevention and treatment of diseases requiring neurostable, such as depression, anxiety, sleep disorders, attention deficit / hyperactivity disorder (ADHD) and the like.
  • diseases requiring neurostable such as depression, anxiety, sleep disorders, attention deficit / hyperactivity disorder (ADHD) and the like.
  • the present invention is an extracellular polysaccharide produced by Ceriporia laccerata; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or it provides a health functional food having neurostable efficacy containing the extract of the mycelia culture medium as an active ingredient.
  • the health functional food according to the present invention may be in the form of powder, granules, tablets, capsules or beverages, and may be candy, chocolate, beverages, gums, teas, vitamin complexes, health supplements, and the like.
  • the extracellular polysaccharide according to the present invention in the food or the mycelia culture solution comprising the same, dried powder or extract thereof may generally be included in 0.01 to 50% by weight, preferably 0.1 to 20% by weight of the total food weight, In the case of a health beverage composition may be included in a ratio of 0.02 to 10g, preferably 0.3 to 1g based on 100ml.
  • the food is an extracellular polysaccharide produced by the seriporia laccerata of the present invention; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or it may further include a food supplement acceptable food additive with the extract of the mycelia culture medium.
  • the present invention is an extracellular polysaccharide produced by the seriporia lacserata; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or it provides a neurostable method comprising administering the extract of the mycelium culture to a subject in need of neurostable.
  • the subject requiring neurostable may be a mammalian animal, and more specifically, a human.
  • the present invention also provides an extracellular polysaccharide produced by Ceriporia laccerata for use in the preparation of a medicament for neurostable; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or it provides a use of the extract of the mycelium culture.
  • the neurostable method can be used for the prevention and treatment of diseases requiring neurostable, for example, depression, anxiety, sleep disorders, attention deficit / hyperactivity disorder (ADHD).
  • diseases requiring neurostable for example, depression, anxiety, sleep disorders, attention deficit / hyperactivity disorder (ADHD).
  • ADHD attention deficit / hyperactivity disorder
  • the light source After inoculating 600 ml of the PDB culture strain with a starter in a state of cooling to 23 ° C., while ventilating air at 0.5 to 1.5 kgf / cm 2, the light source maintains a blue LED, illuminance of 0.5 LUX, and a concentration of carbon dioxide.
  • the Ceriporia laccerata mycelium was prepared by culturing the Ceriporia laccerata mycelium at 2,000 ppm for 10 days liquid culture at a constant temperature of 23 °C.
  • the dry powder of the Ceriporia laccerata mycelium culture medium was prepared by pulverizing and drying the Ceriporia laccerata mycelium culture medium prepared in Preparation Example 1.1 at 25 ° C. for 72 hours using a vacuum freeze dryer.
  • the EPS prepared in Preparation Example 1 was dissolved in 0.1 M Na 2 SO 4 /0.05 M NaN 3 (adjust the pH to 4 with glacial acetic acid) to 1% (w / v), and then 4,000 rpm. After centrifugation for 0.5 hours, only the supernatant was filtered with a 0.45 ⁇ m syringe filter and analyzed by GPC.
  • GPC analysis conditions were used for the refractive index as a detector, GPC column was used OHpak SB 805 HQ (Shodex, Japan), mobile phase 0.1 M Na 2 SO 4 /0.05 M NaN 3 (with pH 4 as glacial acetic acid) The flow rate of the mobile phase was flowed at 1.0 mL / minute. Standard curves were prepared using Dextran (American Polymer Corporation, USA) with different molecular weights (130 kDa, 400 kDa, 770 kDa, or 1200 kDa), and Knauer K-2310 (refractive index (RI) measuring instrument). Germany) to determine the molecular weight of EPS. The measurement conditions are summarized in Table 1 below.
  • the molecular weight of EPS of the present invention was found to be about 120 kDa.
  • the EPS prepared in Preparation Example 1 was second-purified and treated with proteolytic enzymes to determine sugar and protein content.
  • the first purified EPS (EPS prepared in Preparation Example 1) is dissolved in distilled water and centrifuged at 8,000 rpm for 20 minutes to separate the supernatant, and the amount of the supernatant is 2-3 times the amount of the separated supernatant. Ethanol was added and placed in a 4 ° C. refrigerator for 12 hours. Thereafter, only the supernatant was again centrifuged at 8,000 rpm for 20 minutes in the stationary material, and the precipitate was recovered to obtain a second purified EPS. After dissolving the second purified EPS in distilled water, the proteolytic enzyme alcalase was treated at 50 ° C. for 30 minutes at a concentration of 0.5% (w / v).
  • sugar content was measured by the phenol-sulfuric acid method (phenol-sulfuric acid method). Specifically, 25 ⁇ l of 80% (v / v) phenol was added to 1 mL of the diluted sample, 2.5 mL of sulfuric acid was added, cooled to room temperature, and absorbance was measured at 465 nm to calculate sugar content.
  • phenol-sulfuric acid method phenol-sulfuric acid method
  • protein content was measured by the BCA method (Smith PK et al. , Analytical Biochemistry , 150 (1): 76-85, 1985) and bovine serum albumin was used as a standard.
  • the sugar content and protein content measured as described above are shown in Table 2 below, and the sugar content was found to be 45 to 51 wt% and the protein content was 33 to 34 wt%.
  • EPS mainly contained mannose, galactose and glucose.
  • EPS isolated from Ceriporia laccerata mycelium culture medium intracellular calcium concentration after treatment of EPS prepared in Preparation Example 1 to hippocampal single cells isolated from fetal hippocampal neural tissues of rats The change was measured. Specifically, hippocampal neural tissues were isolated from fetuses of 18 to 19 days pregnant under a dissecting microscope and 0.25% trypsin was added and reacted at 37 ° C. for 10 minutes. Subsequently, the tissue was washed several times with HBSS (Hanks' Balanced Salt Solution) to remove trypsin, and the hippocampal cells were separated into single cells with a 1 ml pipette.
  • HBSS Hort' Balanced Salt Solution
  • the intracellular calcium concentration change is shown in Table 3 and FIG.
  • EPS according to the present invention is a calcium channel while acting on the flow channel in the receptor to the low affinity antagonists (Ca + 2 channel) suppressed and the calcium ion (Ca 2+) of the glutamate receptor activity by significantly reducing the inflow cells of the active Reduced.
  • the results show that the EPS according to the present invention has a neurostable effect through inhibition of glutamate (exciting neurotransmitter) receptors.

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PCT/KR2015/011718 2014-11-03 2015-11-03 세리포리아 락세라타에 의해 생산되는 세포외다당체를 유효성분으로 함유하는 신경안정용 조성물 WO2016072710A1 (ko)

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US10226494B2 (en) 2014-11-27 2019-03-12 Fugenbio Co., Ltd. Antioxidant composition containing extracellular polysaccharide produced using Ceriporia lacerata as active ingredient

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KR101645652B1 (ko) * 2014-11-03 2016-08-08 (주)퓨젠바이오농업회사법인 세리포리아 락세라타에 의해 생산되는 세포외다당체를 유효성분으로 함유하는 성기능개선용 조성물

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