Remedies

专利摘要:
The present invention requires a growth factor production inducing action, characterized in that it contains an acid polysaccharide having a growth factor production inducing action, its degradation products, acid oligosaccharides, acidic monosaccharides, acidic sugar alcohols and salts thereof. Therapeutic or prophylactic agents of patients; Food, beverage or feed for inducing growth factor production; Cosmetics for inducing growth factor production; Provide growth factor production regulators.
公开号:KR20020004997A
申请号:KR1020017012954
申请日:2000-04-14
公开日:2002-01-16
发明作者:사가와히로아끼；사까이다께시；고바야시에이지；리투오-핑；오오노기히로무；니시무라가오리；니시야마에이지；우후아-캉；미즈따니시게또시；가또이꾸노신
申请人:오미야 히사시；다까라 슈조 가부시키가이샤；
IPC主号:

专利说明:

Remedy {REMEDIES}
[2] As acidic polysaccharides derived from seaweeds, sulfated polysaccharides such as rhamnan sulfuric acid derived from green algae, sulfated galactan derived from red algae, and sulfated fucose-containing polysaccharide derived from brown algae are known. For example, fucoidan is a sulfated fucose-containing polysaccharide contained in brown algae, echinoderm and the like, and contains sulfated fucose as a constituent sugar. Shark cartilage and the like also contain sulfated polysaccharides.
[3] As the physiological action of sulfated polysaccharides such as fucoidan, cancer growth inhibiting activity, cancer metastasis inhibiting activity, anticoagulant activity, antiviral activity and the like are known, and the use as a medicine is expected to be developed.
[4] Hepaline, heparin sulfate, low molecular weight heparin with an average molecular weight of 4400-5600 are known as a substance having a hepatocyte proliferation factor production action (Japanese Patent Laid-Open No. 6-312941), but other sulfated polysaccharides such as fucoidan and synthetic There is no report on the induction of growth factor production such as sulfated polysaccharide.
[1] The present invention relates to the use of an acidic sugar compound having a physiological activity as a medicine, food, beverage, feed or cosmetics.
[25] BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the DEAE-cellulose A-800 column elution pattern of Kagome kelp derived fucoidan.
[26] 2 is a diagram showing a calibration curve of sulfuric acid content using sodium sulfate solution as a standard sample.
[27] Best Mode for Carrying Out the Invention
[28] In the present invention, the acidic polysaccharide having a growth factor production inducing action may be a growth factor production inducing action, and is not particularly limited, but is not limited to acidic polysaccharides such as alginic acid, pectin, pectinic acid, hyaluronic acid, chondroitin sulfate, keratan sulfate, Sulfated polysaccharides such as delta-martan sulfate, sulfated polysaccharides derived from animals, such as sulfated polysaccharides derived from dermal animals, sulfated polysaccharides derived from fish, such as sulfated polysaccharides derived from shark cartilage, acidic polysaccharides derived from plants, such as wormwood Sulfated polysaccharide, vine sulfated polysaccharide, aloe sulfated polysaccharide, chrysanthemum leaf sulfated polysaccharide, microbial sulfated polysaccharide, such as Chlorella sulfated polysaccharide, Spirulina Sulfated polysaccharide derived from, Sulfated polysaccharide derived from algae, etc. can be used preferably.
[29] Sulfated polysaccharides derived from algae include lambnan sulfates derived from algae, sulfated galactans derived from red algae, such as woodworms, vulgaris, giant kelp, pterochlordiacarirea, carrageenan, agar, agarose, Sulfated fucose-containing polysaccharides derived from agalopectin, porpyran, and brown algae such as fucoidans, sulfated fucoidans, sulfated fucogluclonomannans, gluclooxylofencans, sarugatonic acid, gluronomannogalactan, xyllofuco Gluclonan, ascoryran, gluclonogalactofcan, sulfated gluclonovofan and the like can be used. In particular, fucoidan, sulfated fucogalactan, λ-carrageenan, chondroitin sulfate B, chondroitin sulfate D, alginic acid, agalopectin and the like can be preferably used in the present invention. Acidic polysaccharides derived from cyanobacteria such as sulfated polysaccharides derived from spirulina and acidic polysaccharides derived from green algae such as sulfated polysaccharides derived from chlorella can be used. In particular, the sulfated polysaccharide derived from spirulina is useful for improving liver function by inducing its hepatocyte proliferation factor production, and has a remarkable effect, for example, in improving symptoms of hepatitis C. Phosphoric acid polysaccharides such as nucleic acids are also included in the acidic polysaccharide of the present invention.
[30] The sulfated fucose-containing polysaccharide used in the present invention is preferably exemplified by algae-derived fucoidan, and is not particularly limited as long as it has a growth factor production-inducing action with a polysaccharide having sulfated fucose as a component. Instead of eukaryotic animals such as sea cucumbers, sea urchins and starfish, fucoidans may be used.
[31] These can be used individually or in mixture of 2 or more types. Moreover, as long as the degradation products and salts of these illustrated acidic polysaccharides exhibit growth factor production inducing action, they can be used without particular limitation.
[32] What is necessary is just to prepare these acidic polysaccharides by a well-known method, respectively, and can use a purified product or its acidic polysaccharide content, etc. for this invention. As the acidic polysaccharide content, sulfated polysaccharide fraction can be preferably used, and as this fraction, sulfated polysaccharide fraction derived from algae and sulfated polysaccharide fraction derived from shark cartilage can be preferably used. In addition, algae, sea cucumbers, shark cartilage and the like can be used as raw materials for the sulfated polysaccharide-containing material. For example, kagome kelp, seaweed kelp, toro kelp, fucus, big sea horse, Okinawa big sea horse, seaweed, black horse, rhubarb, Ecklonia, resonia nigre Seaweeds such as kelp, Nagamotsumo, and banyan are particularly preferred for use in the present invention because they are rich in fucoidan for use in the present invention, such as sensium nigrescens and Ascophyllum nodosum.
[33] The synthetic sulfated polysaccharide used in the present invention may be any one having a growth factor production inducing action, and is not particularly limited. However, the use of sulfated polysaccharide which has been used as a medicine is preferred. Examples of this synthetic sulfated polysaccharide include dextran sodium sulfate. This compound is the sodium salt of the sulfated ester obtained by sulfated partial decomposition of dextran produced by fermentation of sucrose by Leuconostoc mesenteroides van Tieghem.
[34] In the present invention, synthetic sulfated polysaccharides such as sulfated starch, sulfated cardan and sulfated pectin can be used, and highly sulfurized sulfated polysaccharide obtained by sulfated sulfated polysaccharide can be preferably used.
[35] The position of the sulfate group of the sulfated polysaccharide used in the present invention is not particularly limited as long as the growth factor production-inducing action is expressed, but the sulfated polysaccharide, fucoidan, lambda -carrageenan, and chondroitin sulfate D in which the second position of the constituent sugar is sulfated And these decomposition products can be preferably used in the present invention. In addition, the sulfuric acid content (or the number of sulfate groups) of the sulfated polysaccharide is not particularly limited as long as the growth factor production-inducing action is expressed. The decomposition products of acidic polysaccharides include oligosaccharides and monosaccharides, and oligosaccharides having a second-position sulfuric acid group, monosaccharides such as fucose-2-sulfuric acid and glucose-2-sulfuric acid can be used. These sulfated monosaccharides, sulfated oligosaccharides, and sulfated polysaccharides may be prepared by their general synthetic methods, and preparations and purified products may also be used in the present invention. In the present invention, oligosaccharides are defined as sugar compounds in which monosaccharides are linked in a range of 2 to 10, and polysaccharides are defined as sugar compounds in which at least 11 monosaccharides are linked.
[36] For example, fucoidan is prepared from kelp kelp, and the fucoidan can be separated into glucuronic acid-containing fucoidan (called U-fucoidan) and glucuronic acid-free fucoidan (called F-fucoidan), and the active ingredient of the present invention As each fucoidan can be used. Moreover, sulfated fucogalactan can be prepared and used from kagome kelp.
[37] Moreover, agalopectin can be prepared and used from agar.
[38] U-fucoidan and F-fucoidan are prepared by preparing fucoidan from kagome kelp, and then separated using anion exchange resin, surfactant, and the like. The abundance ratio of U-fucoidan and F-fucoidan derived from Kagome kelp is about 1: 2, U-fucoidan contains fucose, mannose, galactose, glucuronic acid, sulfuric acid content is about 20%, F-fucoidan Silver contains fucose and galactose, has a sulfuric acid content of about 50%, and a molecular weight is distributed around about 200,000 in both substances (18th Glucose Symposium Summary, p. 159, 1996).
[39] For example, a fucoidan solution prepared from Kagome Kelp can be separated into U-fucoidan and F-fucoidan by applying to a DEAE-cellulose A-800 column and then eluting with NaCl-containing buffer by concentration gradient method. An example thereof is shown in FIG. 1. That is, FIG. 1 is a diagram showing separation between U-fucoidan and F-fucoidan, in which the front peak is U-fucoidan and the rear peak is F-fucoidan.
[40] Moreover, for example, the sulfated polysaccharide derived from Locust fern, the sulfated polysaccharide derived from Lili, the sulfated polysaccharide derived from Pterochromia, the sulfated polysaccharide derived from other algae, the fucoidan derived from Hatban, the fucoidan derived from the large thread, Okinawa and the big silk derived Fucoidan, Fucoidan derived from seaweed, Fucoidan derived from Resonia, Fucoidan derived from Ascofilum, and Fucoidan derived from other algae can also be prepared by a known method and used in the present invention.
[41] Examples of sea cucumbers containing fucoidan include sea cucumbers described in Japanese Patent Application Laid-open No. Hei 4-91027, and fucoidans can be prepared from sea cucumbers by the method described in this publication.
[42] Further, decomposition products of acidic polysaccharides, such as sulfated polysaccharides and fucoidan decomposition products, which have a growth factor production-inducing action of the present invention, are prepared by known methods such as enzymatic methods, chemical methods, physical methods, and the like. Degradates with factor production induction can be selected and used.
[43] The decomposition products vary depending on the acidic polysaccharide to be decomposed, but the molecular weight is preferably about 100,000 to 200, more preferably 30,000 to 1000, obtained by decomposing the acidic polysaccharide.
[44] A preferred method for preparing the decomposed product of the acidic polysaccharide used in the present invention is an acid decomposition method, and by decomposing the acidic polysaccharide, a decomposed product having a growth factor production inducing action can be prepared.
[45] The acid decomposition condition of the acidic polysaccharide used in the present invention is not particularly limited as long as it is a condition under which a decomposition product having a growth factor production inducing action (hereinafter referred to as the decomposition product of the present invention) is produced.
[46] For example, the decomposition product of the present invention is produced by dissolving or suspending the acidic polysaccharide in an acidic aqueous solution or the like. In addition, by heating at the time of reaction, the time required for producing the decomposition product of the present invention is shortened.
[47] The type of acid that dissolves or suspends the acidic polysaccharide is not particularly limited, but may include inorganic salts such as hydrochloric acid, sulfuric acid, and nitric acid, organic acids such as citric acid, formic acid, acetic acid, lactic acid, and ascorbic acid, or cation exchange resins, cation exchange fibers, and cations. Solid acids such as exchange membranes can be used.
[48] The concentration of the acid is also not particularly limited, but is preferably used at a concentration of about 0.0001 to 5 N, more preferably about 0.01 to 1 N. Moreover, reaction temperature is not specifically limited, either, Preferably it is 0-200 degreeC, More preferably, you may set to 20-130 degreeC.
[49] Moreover, reaction time is not specifically limited, either, Preferably it is set to several seconds-several days. What is necessary is just to select suitably the kind and concentration of an acid, reaction temperature, and reaction time according to the production | generation amount of the decomposition product used for this invention, and the polymerization degree of a decomposition product. For example, in the production of a decomposition product of fucoidan, organic acids such as citric acid, lactic acid and malic acid are used, and the acid concentration is several 10 mM to several M, the heating temperature is 50 to 110 ° C, preferably 70 to 95 ° C, and heating. The decomposition product of the present invention can be prepared by appropriately selecting the time in the range of several minutes to 24 hours. Examples of fucoidan acid decomposing products include acid decomposing products of fucoidan derived from Kagome kelp, which can be used as plant fibers having a neophysiological function which is highly effective in inducing growth factor production, particularly inducing hepatocyte proliferation factor production.
[50] The degradation products of the present invention can be fractionated as an indicator of the growth factor production induction action, for example, the acid degradation products can be molecular weight fractionated by gel filtration, fractionation by molecular weight fractionation membrane and the like.
[51] As an example of the gel filtration method, arbitrary molecular weight fractions, such as molecular weight more than 25000, molecular weight 25000-10000, molecular weight 10000-5000, molecular weight 5000 or less, can be prepared using cellulose GCL-300, Using fine GCL-25, for example, a fraction having a molecular weight of 5000 or less is prepared in an arbitrary molecular weight fraction such as molecular weight of 5000 to 3000, molecular weight of 3000 to 2000, molecular weight of 2000 to 1000, molecular weight of 1000 to 500, molecular weight of 500 or less. can do.
[52] Moreover, molecular weight fractionation can be industrially performed using an ultrafiltration membrane. For example, using fraction FE10-FUSO382 manufactured by Daiseru Co., Ltd., a fraction having a molecular weight of 30000 or less and a fraction having a molecular weight of 6000 or less can be obtained by using the same FE-FUS-T653. Can be prepared. Moreover, the fraction of molecular weight 500 or less can also be obtained by using a nanofilter membrane, and arbitrary molecular weight fractions can be prepared by combining these gel filtration methods and the molecular weight fractionation method.
[53] As the decomposition products of acidic polysaccharides, such as fucoidans, which have a growth factor production inducing action which can be used in the present invention, compounds represented by the formula (I) and compounds represented by the formula (II) are exemplified. / 26896 pamphlet, international publication 99/41288 pamphlet can be prepared. Sulfated polysaccharides and oligosaccharides having a repeating structure of the compound represented by the formula (I) can also be used as the sulfated polysaccharide having the growth factor production inducing action of the present invention.
[54] The compound represented by the formula (I) is treated with the aforementioned F-fucoidan with an endo-sulfated polysaccharide degrading enzyme (F-fucoidan specific degrading enzyme) produced by Altereromonas species SN-1009 (FERM BP-5747), It can obtain by refine | purifying from the decomposition product. About the content and site | part of the sulfuric acid group in this compound, arbitrary things can be refine | purified among the decomposition products. This decomposed product also contains a multimer of the compound represented by the formula (I), and can be separated and purified according to the purpose.
[55] The compound represented by the formula (II) is treated with the aforementioned U-fucoidan with an endo sulfate polysaccharide degrading enzyme (U-fucoidan specific degrading enzyme) produced by Flavobacterium sp. SA-0082 (FERM BP-5402), It can obtain by refine | purifying from the decomposition product. About the content and site | part of the sulfuric acid group in this compound, arbitrary things can be refine | purified among the decomposition products. In addition, the decomposed product also contains a multimer thereof having the compound represented by the formula (II) as a basic skeleton, and can be separated and purified according to the purpose.
[56] Examples of the compound represented by the formula (I) include a compound represented by the formula (VI) described later. Examples of the compound represented by the formula (II) include a compound represented by the formula (VII) described later.
[57] Furthermore, a polymer of glucuronic acid and mannose can be obtained by heat treatment of fucoidan derived from Kagome kelp in the presence of an organic acid, and this polymer can also be used as an acidic polysaccharide having an effect of inducing the growth factor production of the present invention. Moreover, the polymer of arbitrary polymerization degrees can be prepared by adjusting heat processing conditions and a heat time.
[58] Acidic polysaccharides having a growth factor production-inducing action in the present invention include synthetic sulfated polysaccharides and include cellulose, starch, mannan, xylene, alginic acid, pectin, pectinic acid, fructan, arabinane, chitin, pullulan Sulfur oxides, such as xyloglucan, dextran, and starch, can be used. For example, synthetic sulfated alkyl polysaccharides such as synthetic sulfated polysaccharides such as riboplanan sulfuric acid, xycloplanan sulfuric acid, lentinan sulfuric acid, gadran sulfuric acid, and mannopyranan sulfuric acid, and riboplanan sulfuric acid having palmitoyl group can be used. Can be used. In addition, by sulfiding the sulfated polysaccharide or its degradation product, a highly sulfurized sulfated polysaccharide or a highly sulfated decomposition product can be prepared. These sulfated polysaccharides, highly sulfurized sulfated polysaccharides, and high sulfur oxide decomposition products may be prepared by known methods, respectively, and the decomposition products may also be prepared by known methods and used in the present invention. Moreover, commercially available dextran sulfuric acid and sulfated cellulose can be used, and salts, such as those synthetic sulfated polysaccharides, etc. may be used.
[59] The acidic oligosaccharide is preferably sulfated oligosaccharide, and the acidic monosaccharide is preferably sulfated monosaccharide, and specific examples thereof are the same as those described above. Such sulfated oligosaccharides or sulfated monosaccharides can be prepared by sulfated corresponding oligosaccharides and monosaccharides by known methods, respectively. Moreover, these salts can also be used preferably. In addition, sulfated polysaccharides, sulfated oligosaccharides, and fatty acid derivatives of sulfated monosaccharides are also included in the sulfated polysaccharides, sulfated oligosaccharides, and sulfated monosaccharides of the present invention. These can be used individually or in mixture of 2 or more types, respectively.
[60] In the present invention, a growth factor for inducing production is not particularly limited as long as it has an activity of promoting cell growth. Hepatocyte growth factor (HGF), nerve growth factor (NGF), neurotrophic factor, epidermal growth factor, milk derived Growth factor, fibroblast growth factor, brain-derived fibroblast growth factor, acidic fibroblast growth factor, platelet-derived growth factor, platelet basic protein, connective tissue activating peptide, insulin-like growth factor (IGF), colony-forming stimulating factor, erythropoie Ethyne, thrombopoietin, T cell growth factor, interleukins (eg interleukin 2, 3, 4, 5, 7, 9, 11, 15), B cell growth factor, cartilage derived factor, cartilage derived growth factor, bone derived growth Factor, skeletal growth factor, endothelial cell growth factor, endothelial cell growth factor, eye-derived growth factor, testis-derived growth factor, Sertoli's cell-derived growth factor, mammary gland stimulation factor, spinal cord Growth factor, macrofuge-derived growth factor, recycled mesenchymal growth factor, transforming growth factor-α, transforming growth factor-β, heparin-binding EGF-like growth factor, amphilegulin, SDGF, betacellulin, epilegulin Neuroneutrophils derived from neutrophil 1, 2, 3, vascular endothelial growth factor, neurotropin, BDNF, NT-3, NT-4, NT-5, NT-6, NT-7, and glial cell line , Hepatocellular factor, midkine, pleurotropin, ephrin, angiopoietin, activin, tumor necrosis factor, interferon and the like.
[61] Among them, at least one selected from the group consisting of HGF, NGF and IGF from the viewpoint of preventing and treating liver disease, preventing and treating neurological diseases, and preventing and treating diabetes, uses the active ingredient according to the present invention. It is preferable to induce production.
[62] HGF exhibits hepatic cell proliferation, protein synthesis promoting action, cholestasis improvement, and further prevention of renal failure caused by drugs. HGF mRNA is also synthesized in the brain, kidney, lung, and the like, and is a mesodermal cell growth factor that has proliferative activity in hepatic parenchymal cells, renal tubule cells, and epidermal cells. Thus, by inducing the production of hepatocyte proliferation factors, hepatitis, severe hepatitis, extreme hepatitis, cirrhosis and intrahepatic cholestasis, chronic nephritis, pneumonia, wounds can be treated or prevented.
[63] IGF has a variety of physiological effects on many cells. By inducing the production of IGF, it is possible to treat or prevent type II diabetes (insulin-independent) or growth intestinal disease (small authentication).
[64] NGF is an endogenous growth factor that maintains the survival and function of nerve cells, or elongates nerve cells according to the concentration gradient of NGF, and induces the production of NGF, thereby causing dementia, peripheral neuropathy, cerebrovascular disorders such as Alzheimer's disease, Treatment or prevention of diseases that require repair and regeneration of nerve function caused by brain tumors, cerebral spinal cord, head trauma degenerative diseases, anesthetic drug poisoning, and the like. In addition, the therapeutic or preventive agent of the present invention exhibits the effect of inducing the production of neurotrophic factors, and the therapeutic or preventive agent of the present invention also exhibits the effect of inducing the production of NGF-neurotrophic factors, such as atrophic lateral sclerosis, drug-induced peripheral neuropathy, and diabetes. It is useful for the treatment and prevention of peripheral neuropathy, Alzheimer's disease, Parkinson's disease, sensory neuropathy, pigmented retinopathy and macular degeneration.
[65] Acidic polysaccharides, degradation products thereof, acidic oligosaccharides, acidic monosaccharides, acidic sugar alcohols, and salts thereof used in the present invention have growth factor production-inducing effects, and these compounds are used as therapeutic ingredients for the treatment of diseases requiring growth factor production or Prophylactic agents can be prepared.
[66] The therapeutic or prophylactic agent for patients in need of inducing growth factor production of the present invention is selected from the acidic polysaccharides, the degradation products thereof, the acidic oligosaccharides, the acidic monosaccharides, the acidic sugar alcohols, and the salts thereof used in the present invention. What is necessary is just to formulate this in combination with a well-known medical carrier. The preparation of this formulation is generally carried out with a liquid or solid carrier which can be pharmaceutically acceptable, selected from the acidic polysaccharides, degradation products thereof, acidic oligosaccharides, acidic monosaccharides, acidic sugar alcohols, and salts thereof used in the present invention. And, if necessary, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, and the like are added, and solid agents such as tablets, granules, powders, powders, and capsules, ordinary liquids, It can be used as a suspending agent or emulsion. Moreover, before using this, it can be set as the dry product which can be made into a liquid state by addition of a suitable carrier.
[67] The pharmaceutical carrier can be selected according to the above formulation, and in the case of oral preparations, for example, starch, lactose, white sugar, mannitol, carboxymethyl cellulose, corn starch, inorganic salt and the like are used. Moreover, in preparation of an oral preparation, you may mix | blend a binder, a disintegrating agent, surfactant, a lubricating agent, a fluidity promoter, a mating agent, a coloring agent, a fragrance | flavor, etc.
[68] On the other hand, in the case of parenterals, those selected from the acidic polysaccharides, their degradation products, the acidic oligosaccharides, the acidic monosaccharides, the acidic sugar alcohols, and salts thereof used in the present invention, which are the active ingredients of the present invention, are used as a diluent according to a conventional method. It is dissolved or suspended in distilled water, physiological saline solution, aqueous glucose solution, injectable vegetable oil, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol, etc. and prepared by adding a fungicide, stabilizer, isotonic agent, analgesic agent, etc. if necessary. do.
[69] The therapeutic or prophylactic agent of the present invention is administered by a suitable route of administration depending on the formulation. The administration method is not particularly limited, either, by content, external use, or injection. Injectables can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermal, and the like, and suppositories include suppositories.
[70] The dosage as a therapeutic or prophylactic agent of the present invention is appropriately set according to the dosage form, the method of administration, the purpose of use, and the age, weight, symptoms, etc. of the patient to be applied thereto, but is not constant but is generally used in the present invention contained in the formulation. The amount of acid polysaccharide, its degradation product, acid oligosaccharide, acid monosaccharide, acid sugar alcohol, and salts thereof is preferably in an amount of 0.01 to 2000 mg / kg per adult. Of course, since the dosage varies according to various conditions as described above, an amount smaller than the dosage may be sufficient in some cases, or may be necessary beyond the range. In the case of oral preparations, the therapeutic or prophylactic agent of the present invention may be orally administered as it is within the desired dosage range, and may be added to any food or drink and consumed daily. Moreover, what is chosen from the acidic polysaccharide used for this invention, its degradation product, the acidic oligosaccharide, the acidic monosaccharide, the acidic sugar alcohol, and its salt may be used as a raw material of the food-drinks for inducing growth factor production.
[71] The soy sauce that has undergone partial resection is quickly regenerated to its original size. Although the body of this hepatic regeneration factor has not been identified for a long time, HGF was found in the plasma of extreme hepatitis patients and isolated and purified from the plasma of the patient (J. Clin. Invest., 88 414-419, 1988). In addition, cDNA of human HGF was also cloned, and the primary structure of HGF was also clarified (Biochem. Biophys. Res. Commun., 163 967-973, 1989). It has also been found that scatter factor (SF), which enhances cell motility, and tumor cytotoxic factor (TCF), which are tumor cell disturbance factors, and HGF are the same substance (Proc. Natl. Acad. Sci. USA, 88 7001-7005, 1991: Biochem. Biophys. Res. Commun., 180 1151-1158, 1991).
[72] HGF promotes the proliferation of many epithelial cells such as bile duct epithelial cells, renal urinary tubule epithelial cells, and gastric mucosa cells as well as hepatocytes. In addition, it stimulates epithelial motility, angiogenesis, and morphogenesis seen in the luminal formation of epithelial cells, and HGF is a multifunctional active substance that exhibits a wide variety of physiological activities. That is, in various organs, it promotes the proliferation of epithelial cells at the time of repairing the disorder of the organ, induces morphogenesis such as hypermotility and angiogenesis.
[73] HGF shows hepatocyte proliferation, protein synthesis promoting action, cholestasis improvement, and further prevention of renal failure caused by drugs. From these facts, it is expected to be a therapeutic drug for severe hepatitis, cirrhosis and intrahepatic cholestasis. However, it has not yet been practical to use HGF itself as a therapeutic drug. In addition, a method of introducing a gene of HGF in gene therapy has been attempted, but it is still far from practical use due to side effects caused by unnecessary time and place. As such, if HGF is not administered externally but can be induced arbitrarily, it is thought to be effective for the treatment and prevention of diseases requiring enhancement of HGF expression such as hepatitis, cirrhosis, intrahepatic bile ducts, and the like. 1, prostaglandin E ₁ , E ₂ , heparin and the like has been confirmed to induce action. IL-1, prostaglandins E ₁ , E ₂ induce the production of HGF by inducing transcription of the HGF gene.
[74] On the other hand, heparin is known to induce HGF production, but it does not induce the transcription of HGF gene, but promotes the production of HGF by promoting the post-translational step of mRNA. In other words, in the state where HGF gene transcription is not induced, There is no effect of inducing HGF production. In contrast, induction of transcription of the HGF gene shows significant production induction.
[75] In addition, the active ingredient according to the present invention does not necessarily directly induce the transcription of growth factors such as HGF, but when their transcription is induced, it significantly promotes such transcription, and also after the transcription such as translation. It is estimated to be able to promote, and consequently has an action of inducing an increase in the production of growth factors. That is, the "growth factor production inducing action" as used in the present invention means the action of inducing the enhancement of the growth factor production, such action is, for example, by the growth factor growth before and after administration of the active ingredient to humans To judge. Here, when "transcription is induced", the above-mentioned active ingredient acts at the time when HGF transcription is required, for example, the transcription is further promoted at an early stage when HGF transcription is promoted, and then there is excess HGF. It is not produced, which means that production of HGF is enhanced when necessary in the body, thereby inducing production of HGF very safely.
[76] The therapeutic or preventive agent of the present invention may further contain a substance which synergistically increases the growth factor production-inducing action of the acidic polysaccharide, its degradation product, the acidic oligosaccharide, the acidic monosaccharide, the acidic sugar alcohol, or salts thereof used in the present invention. .
[77] The "synergistically increasing substance" as used in the present invention means that when the active ingredient according to the present invention is used in combination with this substance, transcription induction is actively performed by this substance, and as a result, growth factor production of the active ingredient according to the present invention is induced. The action is synergistically increased.
[78] As an acid polysaccharide, an acid oligosaccharide, an acid oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, or a substance which synergistically increases the growth factor production-inducing action of these salts used in the present invention, such an acidic polysaccharide, its degradation product, an acidic oligosaccharide, an acidic monosaccharide, The substance having a function of synergistically increasing the growth factor production inducing action of acidic sugar alcohols or salts thereof is not particularly limited, and examples thereof include cytokines, prostaglandins, compounds having a cyclopentene ring, minoxidil, and calpronium chloride. Substances selected from are exemplified. In addition, gingerol, ginger and the like contained in ginger and the like, and curcumin contained in the turmeric and the like are also substances which increase the action of inducing HGF production, and the acidic polysaccharides used in the present invention, its degradation products, acidic oligosaccharides and acidic monosaccharides , Acidic sugar alcohols, or salts thereof can be used as substances that synergistically increase the action of HGF production.
[79] Examples of the cytokines include IL-1 and the like, and prostaglandins include the prostaglandins E ₁ and E ₂ .
[80] Moreover, as a compound which has a cyclopentene ring, the compound represented by following formula (III), and its derivative (s) are illustrated.
[81] These can be used individually or in mixture of 2 or more types, respectively.
[82] For example, a compound having a cyclopentene ring represented by the following formulas (III) to (V), like prostaglandins E ₁ and E ₂ , can induce the transcription of the HGF gene and can be used in the present invention. Synergy with oligosaccharides, acidic monosaccharides, acidic sugar alcohols, or salts thereof can significantly increase the production of HGF. That is, a substance selected from cytokines, prostaglandins, a compound having a cyclopentene ring, a ginger-derived compound, a turmeric-derived compound having an HGF transcription-inducing action, an acidic polysaccharide used in the present invention, its degradation product, an acidic oligosaccharide, By using a mixture selected from acidic monosaccharides, acidic sugar alcohols, or salts thereof as a mixture, the effect of inducing the growth factor production of the acidic polysaccharides, their degradation products, acidic oligosaccharides, acidic monosaccharides, acidic sugar alcohols, or salts thereof used in the present invention is synergistic. It can be increased to obtain a very high production induction effect of HGF.
[83] Moreover, you may use this mixture as a raw material of food-drinks or feed for inducing growth factor production.
[84]
[85]
[86]
[87] For example, the method for preparing a compound represented by Formula III is pamphlet of International Publication No. 98/13328, the compound represented by Formula IV is published in International Publication No. 98/39291 pamphlet, and the compound represented by Formula V is disclosed in International Publication No. 98/40346. It is described in the brochure, respectively, and can be manufactured by the method as described in these.
[88] The method for producing the compound represented by the formula (III) may be any method, and chemical synthesis method [Carbohydrate Res., Vol. 2478, Vol. 217-222 (1993), Helvetica Chimica Acta 55, 2838-2844 (1972)], and a sugar compound, uronic acid and / or uronic acid derivative containing uronic acid, uronic acid derivatives, uronic acid and / or uronic acid derivatives. The cyclopentenone produced in at least 1 sort (s) of heat processing material chosen from the sugar compound containing containing, and its refined product can also be used. The compound represented by the formula (IV) can be obtained by, for example, reacting the compound represented by the formula (III) with glutathione. The compound represented by the general formula (V) can be obtained by, for example, reacting the compound represented by the general formula (III) with propionic anhydride.
[89] In the therapeutic or prophylactic agent of the present invention, the content of a substance which synergistically increases the growth factor production-inducing action of the acidic polysaccharide, its degradation product, the acidic oligosaccharide, the acidic monosaccharide, the acidic sugar alcohol, or salts thereof used in the present invention is determined. What is necessary is just to be the grade which can act synergistically, and although it does not specifically limit, Usually, it is the quantity which becomes 0.001-2000 mg / kg preferably per adult daily. The substance which synergistically increases the induction action may be formulated together with one selected from the acidic polysaccharide, its degradation product, acidic oligosaccharide, acidic monosaccharide, acidic sugar alcohol, or salts thereof used in the present invention, or may be formulated separately. What is necessary is just to implement the method of formulation and the aspect of administration according to the method as described in this specification, and the effect which this invention which the growth factor production induction synergistically increases increases is acquired.
[90] Acidic polysaccharide, its degradation product, acidic oligosaccharide, acidic monosaccharide, acidic sugar alcohol, or salts thereof used in the present invention also have heparanase inhibitory activity, cancer metastasis inhibitory activity, and angiogenesis inhibitory activity. Therefore, cancer metastasis inhibitors and angiogenesis inhibitors can be prepared and provided as an active ingredient selected from these. In particular, the compound represented by the formula (I) derived from fucoidan has a strong heparanase inhibitory effect and a cancer metastasis inhibiting effect, and the pharmaceutical composition containing this compound as an active ingredient is very useful as a cancer metastasis inhibitor. Moreover, the food-drinks containing this compound are highly evaluated as food-drinks for cancer metastasis suppression and angiogenesis suppression.
[91] Foods, beverages or feeds for inducing growth factor production, comprising those selected from acidic polysaccharides, degradation products thereof, acidic oligosaccharides, acidic monosaccharides, acidic sugar alcohols, or salts thereof, which have a growth factor production inducing effect, Increasing the growth factor production inducing effect, improves the symptoms of a disease that requires induction of growth factor production susceptibility to acidic polysaccharides, its degradation products, acidic oligosaccharides, acidic monosaccharides, acidic sugar alcohols, or salts thereof used in the present invention It is very useful for preventing, or for improving the physical condition as described below.
[92] In the food, drink or feed of the present invention, or the cosmetics described later, "contains" includes the meaning of inclusion, addition and dilution, and the content is a state in which the active ingredient used in the present invention is contained in the food, drink or feed. The addition means a state in which the active ingredient used in the present invention is added to a raw material of food, drink or feed, and the dilution refers to a state in which a raw material of food, drink or feed is added to the active ingredient used in the present invention. I speak.
[93] In addition, further comprising those selected from compounds having a synergistically increasing growth factor production-inducing action, such as cytokines, prostaglandins, and cyclopentene rings, may help improve symptoms, prevent or improve the condition of the disease. It is preferable at the point of giving.
[94] Also in the food-drinks of this invention, the preferable aspect of the substance which synergistically increases the said active ingredient, growth factor, or growth factor production induction action is the same as that of the said therapeutic agent or prevention agent. In particular, the food or drink of the present invention, from the viewpoint of liver disease improvement, neurological disease improvement, diabetes improvement, food or beverage for inducing hepatocyte proliferation factor production, insulin-like growth factor production, or nerve growth factor production induction or Feed is preferred.
[95] The production method of the food or beverage of the present invention is not particularly limited as long as a food or beverage having a growth factor production inducing action is obtained. For example, blending, cooking, processing and the like may be performed according to general foods, and may be prepared by the manufacturing method of such foods or beverages, and the acidic polysaccharides used in the present invention having a growth factor production-inducing action in the prepared foods or beverages, their degradation products. And acid oligosaccharides, acidic monosaccharides, acidic sugar alcohols, and salts thereof may be contained as active ingredients.
[96] The food or beverage of the present invention is not particularly limited, but for example, grain processed products (wheat flour processed products, starch processed products, premix processed products, noodles, macaroni, breads, soybean jams, buckwheat, bran, rice flour, noodles, packaging Rice cakes, etc.), oil-processed products (plastic oils, fried oil, salad oil, mayonnaise, dressing, etc.), soybean-processed products (tofu, miso, natto, etc.), meat products (ham, bacon, press ham, sausages, etc.), fishery products (Frozen chopped fish, fish cake, chikuwa (fish pierced or steamed tube fish cake), hanpen (dried shark's minced fish, etc.), Satsuma crab (carrot, burdock, etc. Deep-fried), Tsumira (fish minced meat with egg, salt, flour, etc., dumplings), suji (cooked fish paste made from bone or shell), fish meat ham, sausage, katsuobushi, fish products, canned fish , Tsuku Knives (Sugar of seafood and vegetable seaweed, side dishes with soy sauce), dairy products (raw milk, cream, yogurt, butter, cheese, condensed milk, milk powder, ice cream, etc.), vegetable and fruit processed products (pastes, jams, pickles) Vegetables, fruit drinks, vegetable drinks, mixed drinks, etc., sweets (chocolate, biscuits, sweets, cakes, rice cakes, rice cakes, etc.), alcoholic beverages (table, Chinese sake, wine, whiskey, shochu, vodka, brandy) , Gym, rum, beer, soft alcoholic beverage, fruit wine, liqueur, etc., favorite drink (green tea, black tea, oolong tea, coffee, soft drink, lactic acid drink, etc.), seasoning (soy sauce, sauce, vinegar, mirin, etc.), canned food · Bottled / simmered packaged foods (beef rice, cooked rice, red bean rice, curry, and various other cooked foods), semi-dried or concentrated foods (river paste, other spreads, soba noodles, udon noodles), dried foods (immediately) Noodles, instant curry, instant coffee, powdered juice, powdered soup, Soybean paste soup, cooked food, cooked drinks, cooked soup, etc.), frozen foods (Sukiyaki, Chawan Mushi, steamed fish with uncooked eggs, fish jelly, shiitake, broth, etc.), grilled eel, hamburger steak, Shumai , Dumplings, various sticks, fruit cocktails, etc.), solid foods, liquid foods (such as soups), agricultural and forestry processed products such as spices, livestock processed products, and fish processed products.
[97] The food or beverage of the present invention contains an acidic polysaccharide having a growth factor production-inducing action, a degradation product thereof, an acidic oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, and salts thereof, and for expressing the physiological function thereof. There is no restriction | limiting in particular in the shape as long as a required amount is contained, The orally ingestible shape in the form of tablets, a granule, a capsule, etc. is also included. Sulfated polysaccharides derived from algae and their degradation products, such as fucoidan and its degradation products, which have a growth factor production-inducing action, are very useful as food or beverage manufacturing materials that combine the physiological and plant fiber functions.
[98] The content of the food or beverage in the food or beverage of the present invention selected from an acidic polysaccharide, a degradation product thereof, an acidic oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, and a salt thereof (active ingredient) having a function of inducing growth factor production in the food or beverage is particularly limited. Although it can select suitably from the viewpoint of the sensory and physiological activity, content of an active ingredient is ^10-9 weight part or more per 100 weight part of foods, for example, Preferably it is ^10-7 weight part, for example, 100 weight of drinks per ^10-9 parts by weight or more, preferably 10 ^-7 to 2 parts by weight of a.
[99] In addition, the present invention can be obtained by ingesting the active ingredient in an amount of 0.01 to 2000 mg / kg per adult, and inducing growth factor production orally.
[100] The present invention also provides a biological feed comprising an acid polysaccharide having a growth factor production-inducing action, a degradation product thereof, an acid oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, and a salt thereof.
[101] Then, there is provided a method for breeding organisms, wherein the feed is administered to the organisms.
[102] There is also provided a biological breeding agent comprising an acidic polysaccharide having a growth factor production-inducing action, a degradation product thereof, an acidic oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, and a salt thereof.
[103] In these inventions, organisms are, for example, farm animals, pet animals, and the like, and examples of farm animals include livestock, laboratory animals, poultry, fish, shellfish or shellfish.
[104] Examples of the feed include a condition improving feed based on the growth factor production inducing action.
[105] Biological breeding solvents include immersion solvents, feed additives, beverage additives.
[106] In these inventions, those selected from acidic polysaccharides, their degradation products, acidic oligosaccharides, acidic monosaccharides, acidic sugar alcohols, and salts thereof having growth factor production-inducing action, can be used for breeding efficiencies of organisms, such as survival rate, fat growth rate, spawning rate, and acid. It has the effect of improving autonomy, weaning rate and the like.
[107] Acidic polysaccharides having a growth factor production inducing action, its degradation products, acidic oligosaccharides, acidic monosaccharides, acidic sugar alcohols, and salts thereof are usually administered in 1 kg body weight of the target organism, preferably 0.01 to 2000 mg per day, In addition, it can be added and mixed in the raw material of the artificial feed, or mixed with the powder raw material of the artificial feed, and then further mixed with other raw materials.
[108] The content in the finally obtained feed for the target organisms, selected from acidic polysaccharides having a function of inducing growth factor, its degradation products, acidic oligosaccharides, acidic monosaccharides, acidic sugar alcohols, and salts thereof, is not particularly limited and may be used depending on the purpose. What is necessary is just a ratio of 0.001-15 weight%. For example, when aiming at improving liver function, the ratio of 0.01-10 weight% is suitable.
[109] Artificial feeds include animal raw materials such as fish meal, casein and squid powder, vegetable raw materials such as soybean meal, wheat flour and starch, microbial raw materials such as feed yeast, animal fats and oils such as cod liver oil and squid liver oil, soybean oil, And artificial blended feeds containing vegetable oils such as rapeseed oil, vitamins, minerals, amino acids, antioxidants, and the like. Moreover, the feed for fish, such as fish meat minchi, is mentioned.
[110] There is no particular limitation on the production method of the feed of the present invention, and the blending may be based on the general feed, acid polysaccharide having a growth factor production inducing action, its degradation products, acid oligosaccharide, acid monosaccharide, acid sugar alcohol, and What is necessary is just to contain the effective amount of what is chosen from these salts.
[111] In addition, acid polysaccharides having a growth factor production-inducing action, their degradation products, acid oligosaccharides, acidic monosaccharides, acidic sugar alcohols, and salts thereof are added directly to the water, seawater, holding tank or breeding area, seawater, etc. It can also be administered by dipping the organism. This dipping method is particularly effective when the feed intake of the target organism is reduced.
[112] The concentration of the acidic polysaccharide, its degradation product, the acidic oligosaccharide, the acidic monosaccharide, the acidic sugar alcohol, and the salts thereof having induction of growth factor production in water or seawater is not particularly limited and may be used according to the purpose. Preferably, the ratio of 0.00001 to 1% by weight is appropriate.
[113] In addition, a beverage containing an acid polysaccharide having a growth factor production-inducing action, a degradation product thereof, an acid oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, and a salt thereof may be ingested into the target organism as a breeding drink.
[114] The concentration of the acid polysaccharide, the degradation product thereof, the acid oligosaccharide, the acid monosaccharide, the acid sugar alcohol, and the salts thereof having a growth factor production-inducing effect in the feed is not particularly limited and may be used depending on the purpose. A ratio of 0.0001 to 1% by weight is suitable.
[115] Biological breeding agents, such as immersion solvents, feed additives, and beverage additives, which are selected from acidic polysaccharides, their degradation products, acidic oligosaccharides, acidic monosaccharides, acidic sugar alcohols, and salts thereof having induction of growth factor production, are What is necessary is just to manufacture by the well-known compounding and manufacturing method.
[116] There are no limitations on the organisms to which the present invention can be applied, but aquaculture animals include live animals such as horses, cattle, pigs, sheep, goats, camels, and yama, laboratory animals such as mice, rats, malmots, rabbits, chickens and ducks. Poultry such as turkey, ostrich, red snapper, stone bream, flatfish, flounder, yellowtail, yellowtail, bushy, tuna, foot pad, sweetfish, salmon and trout, cocksucking, freshwater eel, loach, catfish and other fish, prawn, Shellfish such as black tiger, longevity shrimp, crab, etc., shellfish such as abalone, hermit, scallop, and oyster, and pet animals include dogs and cats, and can be widely applied to land and aquatic animals.
[117] Ingesting a feed containing an acid polysaccharide having a growth factor production-inducing action, a degradation product thereof, an acid oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, and a salt thereof, or an acid polysaccharide having a growth factor production-inducing action thereof By immersing the target organism in a solution containing one selected from decomposition products, acid oligosaccharides, acid monosaccharides, acid sugar alcohols, and salts thereof, the conditions of livestock, laboratory animals, poultry, fish, shellfish, shellfish, pet animals, etc. are improved. As a result, bacterial infections and viral infections of the target organism are prevented or treated, and the symptoms are significantly improved in the infected organism. In addition, the health of the target organism is maintained, and the improvement of the survival rate, growth rate, spawning rate, litter rate, weaning rate, growth rate and the like is remarkable.
[118] In addition, these aquatic animals frequently (1) disease caused by bacterial infection, and due to aquaculture in a limited area, when infectious diseases occur, they are quickly infected and wiped out, and (2) parasitic diseases, nutritional diseases, and environmental diseases. (3) It is easy to cause tumors, and (3) farmed animals in narrow breeding areas are stressful, and body surfaces are scratched in breeding facilities, and bacteria and parasites are easily attached everywhere. (4) Stress There is a problem that the appetite is lowered and the growth is slowed. However, the feed of the present invention significantly reduces the stress of farmed animals in a small area due to the condition improving action, and the body surface of the feed facility is increased. Scratching does not occur, and an appetite becomes active, and a growth rate, a litter rate, a spawn rate, a weaning rate, a disease prevention rate, etc. can be improved remarkably.
[119] Acidic polysaccharides, their degradation products, acidic oligosaccharides, acidic monosaccharides, acidic sugar alcohols, or salts thereof used in the present invention having a growth factor production inducing action are useful as active ingredients of cosmetics, and used in the present invention by the present invention. There is provided a growth factor, such as HGF production-inducing cosmetics, comprising as an active ingredient selected from acidic polysaccharides, their degradation products, acidic oligosaccharides, acidic monosaccharides, acidic sugar alcohols, and salts thereof.
[120] In addition, it is preferable to contain a substance selected from the above compounds which synergistically increase the growth factor production inducing action, such as cytokines, prostaglandins, and compounds having a cyclopentene ring, from the viewpoint of helping the desired effect.
[121] Also in the cosmetic of this invention, the preferable aspect of the substance which synergistically increases the said active ingredient, growth factor, or growth factor production induction action is the same as that of the said therapeutic agent or prevention agent. In particular, the cosmetic of the present invention is preferably a cosmetic for inducing hepatocyte proliferation factor production, insulin-like proliferation factor production, or nerve growth factor production induction from the viewpoint of epithelial cell activation.
[122] Fucoidan and its degradation products are particularly preferred as an active ingredient of the cosmetics. For example, F-fucoidan and / or its degradation products, or growth factor production inducing action using the compound represented by the formula (I) as an active ingredient, for example, HGF production inducing action It can provide a bio cosmetic having. The content of the acidic polysaccharide, its degradation product, the acidic oligosaccharide, the acidic monosaccharide, the acidic sugar alcohol, or a salt thereof in the cosmetic for inducing growth factor production is usually preferably 0.0001 to 20% by weight, more preferably 0.001 to 5% by weight.
[123] The growth factor production-inducing, for example, HGF production-inducing cosmetics of the present invention can be produced according to a conventional method according to a known formulation. The growth factor production-inducing cosmetics of the present invention include, for example, lotions, emulsions, creams, packs, bath solvents, face washes, bath soaps or bath detergents.
[124] In the case of applying the cosmetic of the present invention to a desired amount according to each application form, for example, lotions, for example, when applied to the entire face of a person, preferably 0.01 to 5 g, more preferably for one use When about 0.1-2 g is used, the effect which this invention wants that the epithelial cell is activated and effective for beautiful skin is acquired.
[125] The present invention also provides a growth factor production inducer containing as an active ingredient selected from acidic polysaccharides, its degradation products, acidic oligosaccharides, acidic monosaccharides, acidic sugar alcohols, and salts thereof. It is also useful for the screening of disease drugs related to research and growth factors.
[126] The present invention also provides a growth factor production regulator comprising an acidic polysaccharide, a degradation product thereof, an acidic oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, and a salt thereof as an active ingredient.
[127] The growth factor production inducing agent and growth factor production regulator of the present invention may be prepared by a known formulation method using the above-mentioned effective ingredient. Examples of the growth factor production inducing agent include the above-mentioned therapeutic agents and the like. In addition, the growth factor production regulator of the present invention refers to an agent that promotes the transcription of growth factors at the beginning of transcription induction of growth factors in vivo. By the growth factor production regulator of the present invention, the production of growth factors is enhanced only when the production of growth factors is necessary, and has a significant effect of preventing the production of growth factors from becoming excessive.
[128] Fucoidan and / or its degradation product used in the present invention have a particularly strong growth factor production inducing action, growth factor production adjustment action, and is very useful as an active ingredient for use in the formulation of the present invention.
[129] Heparin, which has been known to induce HGF production in the past, does not promote the transcription of HGF mRNA, but fucoidan and its degradation product further promote the transcription of the mRNA at an early stage where the transcription of HGF mRNA is promoted. In vivo, HGF mRNA is not normally transcribed but is transcribed when necessary. Fucoidans and fucoidan digests, such as 7-12SFd-F, described below, further promote transcription only at the beginning of HGF transcription in vivo, after which HGF is not overproduced and the production of HGF is in the body. It is a very safe HGF production modulator in that it is promoted only when needed.
[130] Therefore, in another embodiment of the present invention, the therapeutic or preventive agent, food and drink, etc. may be used as it is for the purpose of adjusting growth factor production induction. The dosage of the growth factor production regulator is not particularly limited as long as it is possible to adjust the growth factor production, but the dosage of the active ingredient according to the present invention, such as the dosage of the active ingredient to the human body, is preferably 0.01 to 2000. The amount which becomes mg / kg (body weight) is mentioned.
[131] Acidic polysaccharides, such as fucoidan and / or its degradation products, which have a growth factor production-inducing action for use in the present invention, do not show any mortality even when orally administered 1 g / kg in oral administration to rats. Dextran sodium sulfate is also a safe compound. Further, other acidic polysaccharides, degradation products thereof, acidic oligosaccharides, acidic monosaccharides, acidic sugar alcohols, or salts thereof used in the present invention are not confirmed to toxicity even if the physiologically effective amount is administered orally to rats.
[132] In another aspect, the present invention requires a growth factor production induction using the extract selected from mugwort extract, vine medicinal extract, aloe extract, mugwort extract, chlorella extract, and spirulina extract having a growth factor production inducing action as an active ingredient You may provide the therapeutic or preventive agent of the patient.
[133] In addition, even if providing a growth factor production inducing food or feed containing an extract selected from mugwort extract, vine sage extract, aloe extract, mugwort extract, chlorella extract and spirulina extract having an effect of producing growth factor as an active ingredient, do.
[134] In addition, you may provide a growth factor production-inducing cosmetics containing extracts selected from mugwort extracts, vines extracts, aloe extracts, mugwort extracts, chlorella extracts and spirulina extracts having a growth factor production-inducing activity as an active ingredient.
[135] Extraction and purification of the extract from such plants and microorganisms can be carried out by the following known methods. Fruits, seeds, leaves, stems, roots, root stems and the like of microorganisms as raw materials and microorganisms are collected at appropriate times and subjected to a drying process such as air drying or the like, followed by extraction. If the raw material is a juice or sap of the plant can also be used as an extract raw material.
[136] Extraction of the extract containing the said active ingredient from the said dried plant and microorganism is performed as follows by a well-known method. After the raw materials are ground or chopped, it may be carried out by a batch or continuous extraction method using a solvent. As an extraction solvent, water, chloroform or alcohols, such as ethanol, methanol, isopropyl alcohol, ketones, such as acetone methyl ethyl ketone, hydrophilic or lipophilic solvents, such as methyl acetate and ethyl acetate, can be used individually or in mixture. Extraction temperature is usually 0-150 degreeC, Preferably it carries out at 5-120 degreeC.
[137] When the extraction is carried out batchwise, the extraction time is about 10 minutes to 20 days, the amount of solvent is usually 1 to 30 times the weight, preferably 2 to 20 times the weight per dry raw material. The extraction operation may be performed by stirring or a settled fat value, or may be combined. The extraction operation may be repeated two or three times as necessary. Examples of the continuous extraction method include a method using a Soxhlet extractor in which a reflux cooler and a siphon are combined. The amount of the solvent and the extraction time are the same as those of the batch extraction method.
[138] The extract used for this invention also includes the insoluble residue removed by filtration or centrifugation from the crude extract obtained by the said operation. Insoluble residues may also be used as active ingredients.
[139] The purification of the active ingredient from the crude extract may be any purification method of a known plant-derived active ingredient, but it is preferable to use a two-phase solvent separation method, a column chromatography method or the like alone or in combination.
[140] Using the obtained extract as an active ingredient, pharmaceuticals, food and drink, feed, and cosmetics can be prepared according to the purpose. What is necessary is just to implement these manufacture according to the said method concerning 1st-3rd invention of this invention.
[141] The content of the extract in the product for each purpose can be determined by its growth factor production-inducing action, which is generally 0.001 to 100% by weight, more preferably 0.01 to 30% by weight, even more preferably in the product. Is 0.1 to 20% by weight.
[142] Moreover, even if these extracts concerning the present invention are orally administered to rats, their toxicity is not observed.
[143] Hereinafter, although an Example is given and this invention is demonstrated more concretely, this invention is not limited to these description at all. In addition,% with respect to the combination of each component in an Example means weight%.
[5] The present invention finds a new physiological action of various acidic sugar compounds such as acidic polysaccharides such as fucoidan, and its purpose is to induce the production of growth factors of various acidic sugar compounds such as acidic polysaccharides such as fucoidan, especially hepatocyte proliferation. The present invention provides a medicine, food, beverage, feed or cosmetics using factor production inducing action, insulin-like growth factor production inducing action or nerve growth factor production inducing action.
[6] When the present invention is schematically described, the first invention of the present invention is selected from an acidic polysaccharide having a growth factor production-inducing action, a degradation product thereof, an acidic oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, or a salt thereof (however, heparin, It relates to a therapeutic or prophylactic agent for patients in need of inducing growth factor production, characterized in that it contains heparan sulfate) as an active ingredient.
[7] According to a second aspect of the present invention, there is provided a food and beverage for inducing growth factor production comprising an acid polysaccharide having a growth factor production-inducing action, a degradation product thereof, an acid oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, and salts thereof. Hereinafter referred to as food and drink) or feed.
[8] The third invention of the present invention relates to a cosmetic for inducing growth factor production comprising an acid polysaccharide having a growth factor production-inducing action, a degradation product thereof, an acid oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, and a salt thereof. will be.
[9] The fourth invention of the present invention relates to a growth factor production regulator comprising an acid polysaccharide, a degradation product thereof, an acid oligosaccharide, an acid monosaccharide, an acid sugar alcohol, and a salt thereof.
[10] In the present invention, as an acidic polysaccharide having a growth factor production inducing action, preferably a sulfated polysaccharide is exemplified, and as the sulfated polysaccharide, a sulfated polysaccharide derived from algae, a sulfated polysaccharide derived from an animal such as dermal animal derived Sulfated polysaccharides such as sulfated polysaccharides derived from sea cucumbers, sulfated polysaccharides derived from fish, such as sulfated polysaccharides derived from shark cartilage, sulfated polysaccharides derived from microorganisms, sulfated polysaccharides derived from plants, such as sulfated polysaccharides derived from mugwort Synthetic sulfated polysaccharide can be used preferably.
[11] Moreover, as a sulfated polysaccharide derived from algae having a growth factor production-inducing action, ramnan sulfuric acid, sulfated galactan, or sulfated fucose-containing polysaccharide can be preferably used. Examples of the synthetic sulfated polysaccharide include dextran sodium sulfate, sulfated starch, sulfate sulfate, pectin sulfate, and the like, and highly sulfurized sulfated polysaccharide obtained by sulfated sulfated polysaccharide can be preferably used. . Moreover, as a sulfated fucose containing polysaccharide, fucoidan can be used preferably. Acidic oligosaccharides are preferably sulfated oligosaccharides, such as sulfated maltose, sulfated lactose, sulfated sucrose, sulfated trehalose, sulfated lactulose, sulfated melibiose, sulfated cellobiose, sulfate Isomaltose, sulfated turanose, sulfated palatinose, sulfated maltotriose, sulfated maltohexaose, sulfated maltoheptaose, sulfated dodecyl-maltohexaose, a compound represented by the following formula (I) or The compound represented by the following formula (II) can be used:
[12]
[13] (Wherein R is OH or OSO ₃ H),
[14]
[15] (Wherein R is OH or OSO ₃ H).
[16] As the acidic monosaccharide, there are preferably sulfated monosaccharides, and for example, sulfated glucose, sulfated galactose, sulfated xylose, sulfated 2-deoxyglucose, sulfated deloose and sulfated mannose can be used. have. As the acidic sugar alcohol, sulfur oxides of sugar alcohols such as sulfated glutitol and the like can also be used. These sulfated oligosaccharides, sulfated monosaccharides and sulfated sugar alcohols may be prepared by these general synthetic methods. The position of the sulfate group and the number of sulfate groups in these sugar compounds are not particularly limited as long as these sulfated oligosaccharides, sulfated monosaccharides, and sulfated sugar alcohols exhibit growth factor production induction.
[17] In this invention, the degradation product of the acidic polysaccharide which has a growth factor production inducing action can also be used. This degradation product also includes a heparin decomposition product having a molecular weight of 4000 or less and a heparan sulfate decomposition product having a growth factor production inducing action.
[18] The substances exemplified by the above acidic polysaccharides, their degradation products, acidic oligosaccharides, acidic monosaccharides and acidic sugar alcohols can be used alone or in combination of two or more thereof, and their salts can also be preferably used.
[19] In the present invention, growth factors include hepatocyte growth factor, insulin-like growth factor, and nerve growth factor.
[20] Production of growth factors for acidic polysaccharides, their degradation products, acidic oligosaccharides, acidic monosaccharides, acidic sugar alcohols, or salts thereof in the therapeutic or prophylactic agent of the first invention of the present invention, the food, beverage or feed of the second invention, and the cosmetic of the third invention. It may further contain a substance which synergistically increases the induction, and examples thereof include cytokines, prostaglandins, compounds having a cyclopentene ring, minoxidil and carpronium chloride.
[21] The food, beverage or feed of the second invention of the present invention is preferably a food, drink or feed for inducing hepatocyte growth factor production, insulin-like growth factor production induction, or nerve growth factor production induction.
[22] The cosmetic of the third invention of the present invention is preferably a cosmetic for inducing hepatocyte growth factor production, insulin-like growth factor production induction, or nerve growth factor production induction.
[23] As cosmetics of 3rd invention of this invention, lotion, emulsion, cream, pack, bath solvent, face wash, bath soap, or bath detergent are illustrated.
[24] "The thing chosen from acidic polysaccharide, its degradation product, acidic oligosaccharide, acidic monosaccharide, acidic sugar alcohol, and these salts" concerning this invention may only be called "active ingredient" in this specification.
[144] Reference Example 1
[145] (1) After sufficiently drying the kagome kelp, 20 kg of the dried product was ground by a free grinding machine (manufactured by Narakikai Seisakusho).
[146] 7.3 kg of calcium chloride dihydrate (made by Nippon Soda Co., Ltd.) was dissolved in 900 liters of tap water, and 20 kg of kagome kelp pulverized products were mixed. It heated up for 40 minutes by steam suction until it became 90 degreeC of liquid temperature from liquid temperature 12 degreeC, and then, it was kept at 90-95 degreeC under stirring for 1 hour, and then cooled and obtained 1100 liters of cabinets.
[147] Subsequently, the solid-liquid separation of the cooled material was performed using the solid-liquid separator (CNA type | mold by West Parrier Co., Ltd.), and about 900 liter of solid-liquid separation supernatant was prepared.
[148] 360 liters of the solid-liquid separated supernatant was concentrated to 20 liters using Daicel Corporation FE10-FC-FUS0382 (fraction molecular weight 30,000). Subsequently, 20 liters of tap water was added, the operation of concentrating to 20 liters was performed 5 times, desalting was performed, and 25 liters of extracts derived from Kagome kelp were prepared.
[149] 1 liter of this extract was lyophilized to obtain 13 g of dried fucoidan derived from kagome kelp.
[150] According to the method described above, kelp-derived fucoidan dried product was prepared from kelp dry ground product. In the same manner, a fucoidan-derived product derived from Lesonia nigressen was prepared from a dry powder of Lesonia nigrescence (trade name Seaweed powder: sold by Andes Boeki Co., Ltd.).
[151] (2) 7 g of the fucoidan dried substance described in Reference Example 1- (1) was dissolved in 700 ml of 20 mM imidazole buffer (pH 8.0) containing 50 mM sodium chloride and 10% ethanol and insoluble by centrifugation. Was removed. DEAE-Cellulopine A-800 column (φ11.4 cm × 48 cm) was equilibrated with the same buffer and centrifuged supernatant, washed with the same buffer and eluted by a concentration gradient of 1.95 M from 50 mM sodium chloride ( 1 fraction: 250 ml). The total equivalent and uronic acid content were calculated | required by the phenol sulfuric acid method and the carbazole sulfuric acid method, and fractions of fractions 43-49, fractions 50-55, and fractions 56-67 were obtained in the elution order. These fractions were then lyophilized after desalting by electrodialysis to obtain I fraction (340 mg) at fractions 43 to 49, II fraction (870 mg) at fractions 50 to 55, and III fraction (2.64 to fractions 56 to 67). g) was prepared respectively.
[152] Fig. 1 shows the DEAE-cellulose A-800 column elution pattern of kagome kelp sulfated fucodan. In Fig. 1, the vertical axis represents the absorbance of 530 nm (black circle in the middle) by the carbazole sulfate method, the absorbance of the 480 nm (white circle in the middle) by the phenol sulfate method, and the conductivity (mS / cm: white square in the middle), and the horizontal axis represents the frag Indicates the option number.
[153] Reference Example 2
[154] (1) Medium 600 which consists of artificial seawater (made by Zamarin Laboratories) pH 8.2 containing Alteromonas spp. SN-1009 (FERM BP-5747), glucose 0.25%, peptone 1.0%, yeast extract 0.05% Inoculated into a 2-liter Erlenmeyer flask, which was dispensed and sterilized (120 占 폚 for 20 minutes), and incubated at 25 占 폚 for 26 hours to obtain a seed culture solution. 20 liters of artificial seawater pH 8.0 containing peptone 1.0%, yeast extract 0.02%, sulfated polysaccharide 0.2% described in Reference Example 2- (2) below, and 0.01% antifoaming agent (KM70 manufactured by Shin-Etsu Chemical Co., Ltd.) Was put into a 30 liter jar fermenter and sterilized at 120 ° C. for 20 minutes. After cooling, 600 ml of the seed culture solution was inoculated and incubated at 24 ° C. for 24 hours under conditions of aeration rate of 10 liters per minute and agitation speed of 250 revolutions per minute. After the completion of the culture, the culture solution was centrifuged to give cells and cultures. The supernatant was obtained. The obtained culture supernatant was concentrated with an ultrafilter equipped with a holofiber of 10,000 molecular weight, and then salted with 85% saturated ammonium sulfate, and the resulting precipitate was collected by centrifugation to contain 1/10 of artificial seawater. It was sufficiently dialyzed against 20 mM tris-hydrochloric acid buffer (pH 8.2) to prepare an endo-sulfated polysaccharide degrading enzyme solution which selectively acts on 600 ml of sulfated polysaccharide.
[155] (2) 2 kg of dried Kagome kelp is crushed by a cutter mill equipped with a screen having a diameter of 1 mm (manufactured by Masyuki Industrial Co., Ltd.), and the resulting kelp chips are suspended in 20 liters of 80% ethanol for 3 hours at 25 ° C. After stirring and filtration with filter paper, the residue was sufficiently washed. The resulting residue was suspended in 20 mM sodium phosphate buffer pH 6.5 containing 40 liters of 50 mM sodium chloride warmed to 95 ° C. and treated at 95 ° C. for 2 hours with stirring several times to extract sulfated polysaccharides.
[156] The suspension in the extract was filtered to prepare a filtrate, and then the filter residue was washed with 3.5 liters of 100 mM sodium chloride to obtain a filtrate again.
[157] After the two filtrates were combined, the temperature was lowered to 30 ° C., and 3000U of alginate (manufactured by Nagase Biochemical Co., Ltd.) was added, followed by 4 liters of ethanol, followed by stirring at 25 ° C. for 24 hours. Subsequently, centrifugation was carried out, and the obtained supernatant was concentrated to 4 liters by an ultrafilter equipped with a holofiber having an exclusion molecular weight of 100,000, and again when the coloring matter was not filtered by 10 mM sodium chloride containing 10% ethanol. Ultrafiltration was continued until.
[158] The precipitate formed in the non-filtrate was removed by centrifugation, the supernatant was cooled to 5 ° C. to pH 2.0 with 0.5 N hydrochloric acid, and then the precipitate, such as protein produced, was removed by centrifugation. The pH was rapidly set to 8.0 with 1 N sodium hydroxide.
[159] Subsequently, ultrafiltration was performed by an ultrafilter equipped with a holofiber having an exclusion molecular weight of 100,000, completely solvent-substituted by 20 mM sodium chloride pH 8.0, centrifugation at pH 8.0 again, and lyophilization. 95 g of sulfated polysaccharide was prepared.
[160] (3) 2 kg of dried kagome kelp was crushed by a cutter mill equipped with a screen having a diameter of 1 mm, and the chips of kelp were suspended in 20 liters of 80% ethanol, stirred at 25 ° C. for 3 hours, and filtered through a filter paper. The residue was washed sufficiently. The residue obtained was 20 containing 30 ml of the endo-sulfated polysaccharide degrading enzyme solution prepared in Reference Example 2- (1), 10% ethanol, 100 mM sodium chloride, 50 mM calcium chloride, and 50 mM imidazole. It was suspended in liter of buffer (pH 8.0) and stirred for 48 hours at 25 ℃. The suspension was filtered through a mesh of 32 μm diameter stainless steel wire and the residue was washed with 10% ethanol containing 50 mM calcium chloride. The residue was further suspended in 10 liters of 10% ethanol containing 50 mM calcium chloride, stirred for 3 hours, filtered and washed with a stainless steel mesh. The residue was suspended under the same conditions, stirred for 16 hours, filtered and washed with a stainless steel wire having a diameter of 32 µm.
[161] The filtrate and washing liquid thus obtained were collected, and ultrafiltered by an ultrafilter equipped with a holofiber having an exclusion molecular weight of 3000, and separated into a filtrate and a non-filtrate.
[162] The filtrate was concentrated to about 3 liters using a rotary evaporator, and then centrifuged to obtain a supernatant. The obtained supernatant was desalted by an electrodialysis machine equipped with a membrane having an exclusion molecular weight of 300, and calcium acetate was added to this solution so as to be 0.1 M, and the resulting precipitate was removed by centrifugation. The supernatant was put into DEAE-cellulose (4 liters of resin) equilibrated with 50 mM calcium acetate in advance and thoroughly washed with 50 mM calcium acetate and 50 mM sodium chloride, followed by a concentration of 50 mM to 800 mM sodium chloride. Eluted by gradient. At this time, the separation amount was 500 ml per piece. The separated fractions were analyzed by cellulose acetate membrane electrophoresis [Analytical Biochemistry, Vol. 37, pp. 197 to 202 (1970)] to find that sulfate was eluted at about 0.4 M. Sugar (near fraction number 63) was uniform.
[163] Therefore, first, the solution of fraction No. 63 is concentrated to 150 ml, and then sodium chloride is added so as to have a concentration of 4 M, and it is put into Phenyl-cellulose (balance of 200 ml of resin) previously equilibrated with 4 M sodium chloride. It was sufficiently washed with M sodium chloride. The nonadsorbable sulfated sugar fraction was collected and desalted by an electrodialysis machine equipped with a membrane having an exclusion molecular weight of 300 to obtain 505 ml of a desalting solution.
[164] 40 ml of the obtained desalting liquid was put into the column (4.1 cm x 87 cm) of cellulose GCL-90 equilibrated with 0.2 M sodium chloride containing 10% ethanol, and gel filtration was performed. Separation was carried out at 9.2 ml per fraction.
[165] Analysis of the total equivalents of all fractions was carried out by the phenol sulfate method (Analytical Chemistry, Vol. 28, pp. 350 (1956)).
[166] As a result, since the sulfated sugar formed one peak, it was desalted by an electrodialysis machine equipped with a membrane having an exclusion molecular weight of 300 at the center of the peak, fraction numbers 63 to 70, and then lyophilized to give 112 mg of the following chemical formula: The dried product of the compound represented by VI was obtained. This compound is hereinafter referred to as 7-12SFd-F.
[167]
[168] (4) 16 mL of 1 M Tris hydrochloric acid buffer solution (pH 7.6) and 16 mL of 1 M CaCl ₂ aqueous solution were added to 80 mL of 2.5% aqueous solution of the III fraction (F-fucoidan) prepared in Reference Example 1- (2). 24 ml of aqueous NaCl solution, 8 ml of the endo-sulfated polysaccharide degrading enzyme solution obtained in Reference Example 2- (1) and 176 ml of distilled water were added, and it heated at 30 degreeC for 3 hours. The enzyme-treated F-fucoidan solution was concentrated with a rotary evaporator so that the final concentration of the enzyme-treated F-fucoidan was 2%, and then dialyzed in distilled water to prepare a 2% enzyme-treated F-fucoidan aqueous solution. This sample was analyzed by HPLC (column: SB802.5, column temperature: 35 ° C, mobile phase: 50 mM NaCl, flow rate: 0.5 ml / min, detection: RI ATT = 8). As a result, it was found that about 40% of the samples were 7-12SFd-F.
[169] Reference Example 3
[170] (1) 2 kg of dried kelp kelp was crushed by a cutter mill (manufactured by Masyuki Industrial Co., Ltd.) equipped with a screen having a hole diameter of 1 mm, and filtered and washed after stirring at 25 ° C. for 3 hours in 80 liters of 80% ethanol. The resulting residue was purified from 50 mM calcium chloride, 100 mM sodium chloride, 10% ethanol, and Alteromonas sp. SN-1009 (FERM BP-5747) endosulfated polysaccharide degrading enzyme prepared in Reference Example 2- (1). It was suspended in 20 liter of 30 mM imidazole buffer (pH 8.2) containing 1U, stirred at 25 ° C for 2 days, and then filtered and washed with a stainless steel mesh having a pore diameter of 32 µm. The obtained residue was suspended in 40 liters of sodium phosphate buffer (pH 6.6) containing 100 mM sodium chloride, 10% ethanol, and 4 g of alginate (manufactured by Nagase Biochemical Co., Ltd.), followed by stirring at 25 ° C. for 4 days, The supernatant was obtained by centrifugation. In order to remove the low molecular weight of alginic acid contained in the obtained supernatant, it concentrated to 2 liters by the ultrafilter equipped with the holographic fiber of the exclusion molecular weight 100,000, and then solution-exchanged with 100 mM sodium chloride containing 10% ethanol. Equivalent amount of 400 mM calcium acetate was added to the solution, followed by stirring, and the supernatant obtained by centrifugation was adjusted to pH 2 with 1 N hydrochloric acid while ice-cooling. The resulting precipitate was removed by centrifugation, and the obtained supernatant was adjusted to pH 8.0 with 1 N sodium hydroxide. The solution was concentrated to 1 liter by ultrafiltration and then solution exchanged with 100 mM sodium chloride. The precipitate produced at this time was removed by centrifugation. In order to remove the hydrophobic substance in the obtained supernatant, sodium chloride was added to the supernatant to 1 M, and it was put into the 3 liter phenyl cellulose cell column (made by the biochemical industry) equilibrated with 1 M sodium chloride, and the waste fraction was collected. The fractions were concentrated by ultrafiltration and lyophilized by solution exchange with 20 mM sodium chloride. The weight of the lyophilisate was 29.3 g.
[171] (2) Endo-type sulfuric acid obtained from this culture by incubating 15 g of the lyophilized product with 400 mM sodium chloride and Flavobacterium sp. SA-0082 (FERM BP-5402) described in pamphlet No. 97/26896. The polysaccharide degrading enzyme was dissolved in 1.5 liter of 50 mM Tris hydrochloric acid buffer containing 9U, reacted at 25 ° C for 6 days, and then concentrated to about 300 ml by an evaporator. The concentrated solution was placed in a dialysis tube of exclusion molecular weight 3500, thoroughly dialyzed, and the remaining liquid in the dialysis tube was added to 4 liters of DEAE-cellulose A-800 equilibrated with 50 mM sodium chloride, thoroughly washed with 50 mM sodium chloride, and then washed 50 to 650. Elution was carried out by a concentration gradient of mM sodium chloride. The same column was also eluted sufficiently with 650 mM sodium chloride. The fraction eluted with 650 mM sodium chloride in the eluted fraction was collected into sulfated fucogalactan fraction, concentrated by ultrafiltration with an exclusion molecular weight of 100,000, and the solution was replaced with 10 mM sodium chloride and lyophilized to freeze the sulfated fucogalactan. 0.85 g of dried product was obtained. The obtained sulfated fucogalactan contained galactose and fucose as constituent sugars, and the molar ratio thereof was about 2: 1.
[172] Reference Example 4
[173] 120 g of sulfated polysaccharide prepared in Reference Example 2- (2) contains 20 mM calcium chloride, 300 mM sodium chloride, 10% ethanol, and endo-sulfated polysaccharide degrading enzyme prepared in Reference Example 2- (1) of 10U. The resultant was suspended in 8 liters of 20 mM imidazole buffer (pH 7.5), stirred at 25 ° C. for 3 days, and ultrafiltered while adding the buffer using an ultrafiltration apparatus equipped with a holofiber having an exclusion molecular weight of 100,000.
[174] The endosulphated polysaccharide degrading enzyme prepared in 34U of Reference Example 3- (2) was added to the ultrafiltration solution, stirred at 25 ° C for 2 days, and subjected to ultrafiltration equipped with a holofiber with an exclusion molecular weight of 100,000. Ultrafiltration with addition.
[175] The filtrate was collected, concentrated to 1.5 liters by an evaporator, then completely desalted by a desalting unit, and equilibrated with 5 mM imidazole hydrochloride buffer (pH 6.5) containing 30 mM sodium chloride in advance. The mixture was washed with 6 liters of the same buffer in an A-800 column, and eluted with a concentration gradient of 30 mM to 50 mM sodium chloride. The amount of liquid required for elution was 48 liters. The eluate was separated by 180 ml and the sugar content was measured by the phenol-sulfuric acid method. Moreover, the absorbance at 232 nm was also measured simultaneously. Since 130 mM to 170 mM sodium chloride eluted fraction formed one peak, these fractions were collected, desalted by a desalting apparatus, and lyophilized to obtain 5.85 g of oligosaccharide. This oligosaccharide was found to be a compound having a molecular weight of 1128 by mass spectrometry and represented by the following chemical formula VII by NMR analysis. Hereinafter, this compound is called 6-2S.
[176]
[177] Reference Example 6
[178] After crushing 1 kg of commercial seaweed dried dried fruit with a cutter mill equipped with a screen with a hole diameter of 1 mm, suspended in 10 liters of 80% ethanol, stirred for 3 hours, filtered and filtered to obtain a residue. The residue was suspended in 20 liters of 40 mM phosphate buffer (pH 6.5) containing 50 mM sodium chloride and treated at 95 ° C. for 2 hours. After cooling the process liquid to 37 degreeC, ethanol was added so that it might become 10%, and after adding 12000U of commercially available alginic acid K (made by Nagase Chemical Co., Ltd.), it stirred at room temperature for 24 hours. The obtained processing liquid was centrifuged, the supernatant was concentrated to 2 liters by the ultrafilter equipped with the holographic fiber of the exclusion molecular weight 100,000, and the produced precipitate was removed by centrifugation. The obtained supernatant was cooled to 5 ° C., 0.5 N hydrochloric acid was added to pH 2.0, and the precipitate formed by stirring for 30 minutes was removed by centrifugation. The pH of the supernatant was 8.0 with 0.5 N sodium hydroxide, and the solution was replaced with 20 mM sodium chloride by ultrafiltration. After adjusting the pH of the solution to 8.0, the supernatant obtained by centrifugation was lyophilized to obtain 90.5 g of Wakame-derived Fucoidan.
[179] Reference Example 7
[180] 1 kg of the dried product of pulverized Fucus vesiculosus was suspended in 10 liters of 80% ethanol, stirred for 3 hours, and filtered through a filter paper to obtain a residue. The residue was suspended in 30 liters of 30 mM phosphate buffer (pH 6.0) containing 100 mM sodium chloride and treated at 95 ° C. for 2 hours. After cooling the process liquid to 37 degreeC, 100 g of activated carbon was added and stirred for 30 minutes. After 3000U of commercially available alginate K was added, ethanol was added to 10% and stirred at room temperature for 24 hours. The obtained processing liquid was centrifuged, the supernatant was concentrated to 2 liters by the ultrafilter equipped with the holographic fiber of the exclusion molecular weight 100,000, and the produced precipitate was removed by centrifugation. The pigment was removed by ultrafiltration while adding the extract to this supernatant. The obtained non-filtrate was cooled to 5 ° C, and then 0.5N hydrochloric acid was added to make the pH 2.0, followed by stirring for 30 minutes to remove the precipitate formed by centrifugation. The pH of the supernatant was 8.0 with 0.5 N sodium hydroxide, and the solution was replaced with 20 mM sodium chloride by ultrafiltration. After adjusting the pH of the solution to 8.0, the supernatant obtained by centrifugation was lyophilized to obtain 71 g of moban-derived fucoidan.
[181] According to the method described above, ascorfilum nordofiber fucoidan was prepared from a dry powder of Ascophyllum nodosum (trade name Algingold: Andean Boeki Co., Ltd.).
[182] Reference Example 8
[183] After dissolving 2 g of kagome kelp-derived fucoidan prepared by the method described in Reference Example 1- (1) in 100 ml of water, adjusting its pH to pH 3 with citric acid, and treating it at 100 ° C. for 3 hours, the acid decomposed product of the fucoidan Was prepared. The hydrolyzate was fractionated by gel filtration with cellulose GCL-300 or cellulose GCL-25 to obtain molecular weight greater than 25,000 (A fraction), 25000 to 10000 (B fraction), and 10000 to 5000 (C fraction). Fraction), more than 5000-2000 (D fraction), more than 2000-500 (E fraction), and 500 or less (F fraction). In addition, these fractions and acid decomposers were desalted and then lyophilized to prepare respective fractions of the acid decomposers and the acid decomposers.
[184] Reference Example 9
[185] 5 kg of commercial salted large horses were mixed with 20 liters of ethanol and chopped finely with scissors. After standing overnight, the residue obtained by filtration with filter paper was suspended in 12.5 liters of water and treated at 95 ° C for 2 hours. After the treatment liquid was filtered through filter paper, 2600 ml of a 2.5% cetylpyrididium chloride solution containing 350 mM sodium chloride was added and left to stand for 3 days. The supernatant was discarded and the precipitate was centrifuged to discard the supernatant. 2.5 liters of 350 mM sodium chloride was added to the obtained precipitate, followed by homogenization and centrifugation. This washing operation was repeated three times. 400 ml of 400 mM sodium chloride was added to the obtained precipitate, homogenized by a homogenizer, ethanol was added to 80%, stirred for 30 minutes, and filtered through a filter paper. 500 ml of saturated sodium chloride 80% ethanol was added to the obtained residue. Then, the mixture was uniformed with a homogenizer, saturated sodium ethanol was added to 1 liter, stirred for 30 minutes, and filtered through a filter paper. This washing operation was repeated until the absorbance of 260 nm of the filtrate became 0 (normally 5 times). After the obtained residue was dissolved in 1.5 liter of 2M sodium chloride, the insolubles were removed by centrifugation and passed through a 100 ml column of DEAE-cellulose A-800 which had previously been equilibrated with 2M sodium chloride. The passage fraction was concentrated to 2 liters by an ultrafilter equipped with a holofiber having an exclusion molecular weight of 100,000, and then the solution was replaced by 2 mM sodium chloride by ultrafiltration. The solution was centrifuged and the obtained supernatant was lyophilized to obtain 22.9 g of large silk derived fucoidan.
[186] Reference Example 10
[187] (1) 50 g of dried loach was finely cut with scissors, suspended in 500 ml of 80% ethanol, and stirred at 25 ° C. for 3 hours, and filtered with a filter paper. The obtained residue was suspended in 30 mM sodium phosphate buffer (pH 6.5) containing 1 liter of 100 mM sodium chloride, treated at 95 ° C. for 2 hours, and then filtered through a stainless steel sieve having a pore diameter of 106 μm. The sodium phosphate buffer solution was added to the obtained filtrate to make 3 liters, 5 g of activated carbon was added, stirred at 25 ° C. overnight, and then centrifuged. The obtained supernatant was concentrated to 200 ml by an ultrafilter equipped with a holofiber having an exclusion molecular weight of 100,000, and then solution exchanged by an ultrafilter to obtain a 10 mM sodium chloride solution. The insolubles in the solution were removed by centrifugation and then lyophilized to obtain 2.3 g of dried product of the sulfated polysaccharide fraction derived from the fern.
[188] (2) In the method described in Reference Example 10- (1), 4.4 g of Sulfate-derived sulfated polysaccharide was prepared from 50 g of a dry grade. Similarly, 1.0 g of sulfated polysaccharide derived from heterocrdia was prepared in a dry heterocrdia capillara.
[189] (3) -1 1 kg of commercially available dry Resonia nigressen powder was suspended in 10 liters of 80% ethanol, stirred at 25 ° C. for 3 hours, and filtered with a filter paper. The obtained residue was suspended in 30 mM sodium phosphate buffer (pH 6.5) containing 20 liters of 100 mM sodium chloride, treated at 95 ° C. for 2 hours, and then filtered through a stainless steel sieve having a pore diameter of 106 μm. 100 g of activated carbon, 2.4 liters of ethanol, and 6,000 U of alginate K were added to the obtained filtrate, and the mixture was stirred at 25 ° C. for 22 hours, followed by centrifugation. The obtained supernatant was concentrated to 1.2 liters with an ultrafilter equipped with a holofiber having an exclusion molecular weight of 100,000, and then the insolubles were removed by centrifugation and left at 5 ° C for 24 hours. The resulting precipitate was removed by centrifugation and the obtained supernatant was solution exchanged with an ultrafilter to give a 100 mM sodium chloride solution. After cooling this solution to 4 degrees C or less, pH was set to 2.0 with hydrochloric acid, and the produced precipitation was removed by centrifugation. The pH of the obtained supernatant was made into 8.0 with sodium hydroxide, and it concentrated to 2 liter, and solution exchanged with 20 mM sodium chloride by the ultrafilter. The insoluble matter in this solution was removed by centrifugation, and then lyophilized to obtain 41 g of dried product of the resonia-derived fucoy fraction.
[190] (3) -2 5 g of the lyophilisate was dissolved in 600 ml of 20 mM imidazole hydrochloride buffer (pH 6) containing 100 mM sodium chloride and previously equilibrated with the same buffer to 5 liters of DEAE-cellulose. It was poured into A-800, washed with 10 liters of the same buffer solution, and eluted with a concentration gradient of 100 to 1600 mM sodium chloride. The amount of liquid used for the elution was 13 liters and the separation was performed at 500 ml per piece. The sodium chloride leaching fractions around 250 mM, 530 mM and 700 mM in the eluted fraction were dialyzed with 500 ml of purified water, respectively, and lyophilized. The lyophilisates were named DEAE 33 fractions, DEAE 37 fractions, and DEAE 40 fractions, respectively, 57 mg. , 24 mg and 62 mg were obtained.
[191] Reference Example 11
[192] 5 kg of sea cucumbers were dismantled to remove the intestines and to collect body walls. 500 ml of acetone per 200 g of body wall wet weight were added, treated with a homogenizer and filtered, and the residue was washed with acetone until no more coloring material was present. The residue was suction dried to give 140 g of dried product. 2.8 liters of 0.4 M saline was added to the dried product, and the mixture was filtered after treatment at 100 ° C. for 1 hour, and the residue was sufficiently washed with 0.4 M saline to obtain 3.7 liters of the extract. 5% cetylpyridium chloride was added to the extract until no precipitation occurred, and the resulting precipitate was collected by centrifugation. The precipitate was suspended in 0.4 M saline solution and centrifuged again, and 1 liter of 4 M saline was added to the obtained precipitate and treated with a homogenizer. Then, 4 liters of ethanol was added while stirring and stirred for 1 hour, followed by filtration. Got it. The precipitate was suspended in 80% ethanol, and the process of filtration was repeated until the absorbance at 260 nm of the supernatant became zero. The obtained precipitate was suspended in 2 liter of 2M saline solution and insolubles were removed by centrifugation. The supernatant was ultrafiltered by an ultrafiltration apparatus equipped with a membrane having a molecular weight of 30,000, completely desalted, and then lyophilized to be 3.7 g. Sea cucumber-derived fucoidan was obtained.
[193] Reference Example 12
[194] 500 mg of agar powder (manufactured by Nakara Lightsk Co., Ltd.) was suspended in 100 ml of distilled water, and then heated to dissolve the agar. Then, it cooled to 45 degreeC and kept warm at 45 degreeC.
[195] To this agar solution, 2 ml of an X50 β-agarase buffer (manufactured by FMC: attached to β agarase) was added, and 100 μl of 1 U / μl β agarase (manufactured by FMC) was added. After the solution was kept at 45 ° C for 24 hours, 2.5 times of ethanol was added thereto, cooled, and centrifuged to recover the precipitate. This precipitate was dried and dissolved in 20 ml of distilled water. The solution was lyophilized to prepare agaroseactin fractions in powder form.
[196] Reference Example 13
[197] (1) 10 g of dry cells of Spirulina platensis were suspended in 100 ml of chloroform and filtered to recover insoluble fractions five times. Thereafter, the procedure of suspending in 100 ml of ethanol and filtering to recover the insoluble fraction was repeated three times. Ethanol was completely removed from the insoluble fraction obtained by this operation and suspended in 100 ml of distilled water. The suspension was kept at 60 ° C. for 1 hour and then centrifuged to obtain supernatant. The supernatant was filtered again, 2.5 times of ethanol was added to the filtrate, cooled at -20 ° C, and centrifuged at low temperature to obtain a precipitate. This precipitate was dissolved in distilled water and lyophilized to prepare a fraction containing a powdered spirulina-derived sulfated polysaccharide.
[198] (2) 20 g of dried spirulina powder (released: Spirulina Research Institute) was added to a homogenizer (manufactured by Nippon Seiki Co., Ltd.), and 400 ml of acetone was added and homogenized at 8000 rpm for 10 minutes. The homogenate was filtered through filter paper to give a residue. The residue was washed three times with acetone in the same manner as described above to obtain an acetone wash residue. The acetone washing residue was washed four times with 90% ethanol and four times with 80% ethanol in the same manner as the acetone washing to obtain an ethanol washing residue.
[199] To the ethanol washing residue was added 30 mM phosphate buffer (pH 7.0) containing 600 ml of 100 mM sodium chloride and 10% ethanol and stirred for 18 hours at room temperature. The mixture was centrifuged at 10000 rpm for 40 minutes to obtain supernatant. Insoluble matters mixed in the supernatant were filtered through filter paper to obtain a crude extract (filtrate). The crude extract obtained was concentrated to 300 ml by an ultrafiltration apparatus equipped with a holofiber having an exclusion molecular weight of 10,000, and then ultrafiltered with 100 mM sodium chloride containing 2 liters of 10% ethanol. Thereafter, the solvent was substituted with 10 mM imidazole hydrochloride buffer (pH 7.0) containing 10% ethanol and 50 mM sodium chloride to obtain 240 ml of spirulina polymer fraction.
[200] Spirulina polymer fraction was added to a DEAE-cellulose fine A-800 column (φ3 × 14.2 cm) equilibrated with 10 mM imidazole-hydrochloric acid buffer (pH 7.0) containing 10% ethanol and 50 mM sodium chloride, 360 ml of the same buffer. The column was washed with and eluted with a concentration gradient of sodium chloride from 0.05 M (200 mL) to 2 M (200 mL). The eluate was partitioned into 10 ml per piece. Fraction No. during elution fraction From 14 to 30, Spirulina sulfated polysaccharide fraction-I (SSP-I), fraction No. From 69 to 77 spirulina sulfated polysaccharide fraction-II (SSP-II), fraction No. From 78 to 83 spirulina sulfated polysaccharide fraction-III (SSP-III), fraction No. 84-99 were named spirulina sulfated polysaccharide fraction-IV (SSP-IV), respectively. SSP-I, SSP-II, SSP-III and SSP-IV were sufficiently dialyzed in distilled water and lyophilized, resulting in 200 mg, 260 mg, 100 mg and 60 mg, respectively.
[201] (3) 10 g of dry cells of Chlorella vulgaris were suspended in 100 ml of chloroform and filtered to recover insoluble fractions three times. Thereafter, the procedure of suspending in 100 ml of ethanol and filtering to recover the insoluble fraction was repeated three times. Ethanol was completely removed from the insoluble fraction obtained by this operation and suspended in 100 ml of distilled water. The suspension was kept at 60 ° C. for 1 hour and then filtered. 2.5 times the amount of ethanol was added to the filtrate, cooled at -20 ° C, and then centrifuged at low temperature to obtain a precipitate. This precipitate was dissolved in distilled water and lyophilized to prepare a fraction containing a sulfated polysaccharide derived from powdery chlorella.
[202] (4) 20 g of dry chlorella powder (release: Chlorella Center Co., Ltd.) was placed in a homogenizer (manufactured by Nippon Seiki Co., Ltd.), and 400 ml of acetone was added and homogenized at 8000 rpm for 10 minutes. The homogenate was filtered through filter paper to give a residue. The residue was washed three times with acetone in the same manner as described above to obtain an acetone wash residue. The acetone washing residue was washed four times with 90% ethanol and four times with 80% ethanol in the same manner as the acetone washing to obtain an ethanol washing residue.
[203] To the ethanol washing residue was added 30 mM phosphate buffer (pH 7.0) containing 600 ml of 100 mM sodium chloride and 10% ethanol and stirred for 18 hours at room temperature. The mixture was centrifuged at 10000 rpm for 40 minutes to obtain supernatant. Insoluble matters mixed in the supernatant were filtered through filter paper to obtain a crude extract (filtrate). The crude extract obtained was concentrated to 310 ml by an ultrafiltration apparatus equipped with a holofiber having an exclusion molecular weight of 10,000, and then ultrafiltered with 100 mM sodium chloride containing 3 liters of 10% ethanol. Thereafter, the solvent was substituted with 10 mM imidazole hydrochloride buffer (pH 7.0) containing 10% ethanol and 50 mM sodium chloride to obtain 203 ml of Chlorella polymer fraction.
[204] Chlorella polymer fraction was added to a DEAE-cellulose fine A-800 column (φ3 × 14.2 cm) equilibrated with 10 mM imidazole-hydrochloric acid buffer (pH 7.0) containing 10% ethanol and 50 mM sodium chloride, and 297 ml of the same buffer. The column was washed with and eluted with a concentration gradient of sodium chloride from 0.05 M (200 mL) to 2 M (200 mL). The eluate was partitioned into 10 ml per piece. Fraction No. during elution fraction From 63 to 68 are named Chlorella Sulfated Polysaccharide Fraction-I (CPS-I), fraction No. From 69 to 75 were named Chlorella Sulfated Polysaccharide Fraction-II (CPS-II). CPS-I and CPS-II were sufficiently dialyzed with distilled water and lyophilized, resulting in 140 mg and 200 mg, respectively.
[205] (5) 10 g of crushed mugwort powder (Altemisia princeps pampan (manufactured by Sakamoto Kanpodo)) was suspended in 100 ml of chloroform and filtered to recover insoluble fractions three times. Thereafter, the procedure of suspending in 100 ml of ethanol and filtering to recover the insoluble fraction was repeated five times. Ethanol was completely removed from the insoluble fraction obtained by this operation and suspended in 100 ml of distilled water. The suspension was kept at 60 ° C. for 1 hour and then filtered. 2.5 times the amount of ethanol was added to the filtrate, and the mixture was cooled at −20 ° C., followed by centrifugation at low temperature to obtain precipitates and wormwood blue fractions. This precipitate was dissolved in distilled water and lyophilized to prepare a fraction containing powdery mugwort-derived sulfated polysaccharide.
[206] (6) 50 g of dried mugwort leaves (released from Sakamoto Kanpodo) were placed in a homogenizer (manufactured by Nippon Seiki Co., Ltd.), and 500 ml of acetone was added to homogenize at 8000 rpm for 10 minutes. The homogenate was filtered through filter paper to give a residue. The above operation was performed twice, and the obtained 100 g of mugwort leaf residue was put into a homogenizer, and 500 ml of acetone was added and homogenized at 8000 rpm for 10 minutes. The homogenate was filtered through filter paper to give a residue. This operation was repeated 4 times to obtain an acetone washing residue. The acetone washing residue was washed four times with 90% ethanol and four times with 80% ethanol in the same manner as the acetone washing to obtain an ethanol washing residue.
[207] To the ethanol washing residue was added 5 liters of 100 mM sodium chloride and 30 mM phosphate buffer (pH 8.0) containing 10% ethanol and stirred for 19 hours at room temperature. The mixture was filtered through filter paper and the crude extract (filtrate) ) The crude extract obtained was concentrated to 2 liters with an ultrafiltration apparatus equipped with a holofiber having an exclusion molecular weight of 10,000, and then ultrafiltered while adding 100 mM sodium chloride containing 10 liters of 10% ethanol. Thereafter, the mixture was concentrated to 500 ml and solvent-substituted with 10 mM imidazole hydrochloride buffer (pH 7.0) containing 10% ethanol and 50 mM sodium chloride. The solution was transferred to a beaker, 1 g of activated carbon was added thereto, stirred at room temperature for 40 minutes, and then centrifuged at 10000 rpm for 40 minutes. Activated carbon incorporated in the supernatant was removed by filtration through filter paper. Thus, 560 mL of mugwort leaf polymer fractions were obtained.
[208] The same buffer solution was added to the DEAE-cellulose A-800 column (φ3.5 x 31 cm) equilibrated with 10 mM imidazole-hydrochloride buffer (pH 7.0) containing 10% ethanol and 50 mM sodium chloride. The column was washed with 940 mL and eluted by a concentration gradient of sodium chloride from 0.05 M (600 mL) to 2 M (600 mL). The eluate was partitioned into 10 ml per piece. Fraction No. during elution fraction From 180 to 202 are named as mugwort leaf acid polysaccharide fraction (YAP), fraction No. From 203 to 270 were named mugwort leaf sulfated polysaccharide fraction (YSP). YAP was sufficiently dialyzed against distilled water and lyophilized to find 250 mg.
[209] To further fractionate mugwort sulfated polysaccharide fractions, mugwort sulfate sulfated polysaccharide fractions were dialyzed with 10 mM imidazole-hydrochloric acid buffer (pH 7.0) containing 3 liters of 10% ethanol and 100 mM sodium chloride. Dialysis sulfated polysaccharide fraction (327 ml) was added to a DEAE-cellulose fine A-800 column (φ3 cm × 14.2 cm) equilibrated with the same buffer. The column was washed with 273 ml of buffer and then 0.1 M ( 200 mL)-2 M (200 mL) was eluted with a concentration gradient of sodium chloride. The eluate was partitioned into 5 ml per piece. Fraction No. during elution fraction From 140 to 154 are named mugwort leaf sulfated polysaccharide fraction-I (YSP-I), fraction No. From 155 to 200 were named mugwort leaf sulfated polysaccharide fraction-II (YSP-II). YSP-I and YSP-II were sufficiently dialyzed with distilled water and lyophilized, respectively, to be 20 mg and 130 mg.
[210] To YSP-II (119.4 mg) 10% ethanol was added 10 mM imidazole-hydrochloric acid buffer (pH 7.0) containing 59.7 mL and 0.2 M sodium chloride and dissolved by stirring overnight at room temperature. The dissolved YSP-II was added to a DEAE-cellulose A-800 column (φ 2.5 cm x 10.2 cm) equilibrated with the same buffer. The column was washed with 200 ml of buffer and eluted with a concentration gradient of 0.2 M (100 ml)-1 M (100 ml) of sodium chloride. The eluate was partitioned into 5 ml per piece. Fraction No. during elution fraction 54 to 70 were named mugwort leaf sulfated polysaccharide fraction-II-2 (YSP-II-2), and fraction No. From 71 to 90 were named mugwort leaf sulfated polysaccharide fraction-II-3 (YSP-II-3), fraction No. 91-120 were named mugwort leaf sulfated polysaccharide fraction-II-4 (YSP-II-4). Lyophilization resulted in 39.5 mg, 61 mg and 57.3 mg, respectively.
[211] (7) Commercially available edible vines were lyophilized by a mixer to obtain vines dried products. The operation of suspending 10 g of dried dried vines in 100 ml of chloroform and filtering to recover insoluble fraction was repeated five times. Thereafter, the operation of suspending in 100 ml of ethanol and filtering to recover the insoluble fraction was repeated three times. Ethanol was completely removed from the insoluble fraction obtained by this operation and suspended in 100 ml of distilled water. The suspension was kept at 60 ° C. for 1 hour and then filtered. 2.5 times the amount of ethanol was added to the filtrate, cooled at -20 ° C, and then centrifuged at low temperature to obtain a precipitate. This precipitate was dissolved in distilled water and lyophilized to prepare a fraction containing powdered sulfated polysaccharide.
[212] (8) Transparent leaf mesophyll portions were recovered from five leaves of commercially available aloe arborescens and lyophilized. 0.481 g of this aloe leaf lyophilisate was suspended in 100 ml of distilled water. The suspension was kept at 60 ° C. for 1 hour and then filtered. 2.5 times the amount of ethanol was added to the filtrate, cooled at -20 ° C, and then centrifuged at low temperature to obtain a precipitate. This precipitate was dissolved in distilled water and lyophilized to prepare a fraction containing powdered aloe leaf meat-derived sulfated polysaccharide.
[213] On the other hand, the green leaf surface portion remaining after the transparent leaf portion was recovered by the above method was pulverized and lyophilized. 3.43 g of the lyophilisate was suspended in 100 ml of chloroform and filtered to recover the insoluble fraction three times. Thereafter, the procedure of suspending in 100 ml of ethanol and filtering to recover the insoluble fraction was repeated three times. Ethanol was completely removed from the insoluble fraction obtained by this operation and suspended in 100 ml of distilled water. The suspension was kept at 60 ° C. for 1 hour and then filtered. 2.5 times the amount of ethanol was added to the filtrate, cooled at -20 ° C, and then centrifuged at low temperature to obtain a precipitate. This precipitate was dissolved in distilled water and lyophilized to prepare a fraction containing powdered aloe leaf surface-derived sulfated polysaccharide.
[214] Reference Example 14
[215] (1) 200 mg (1.1 mmol) of D-(+)-glucose was dissolved in 10 ml of pyridine, and 1.05 g (6.6 mmol) of pyridine sulfotrioxide complex (Pyr · SO ₃ : Tokyo casei) was added at room temperature. Then, it stirred at room temperature and moisture at 60 degreeC for 1 hour. The reaction solution was diluted with water, the pH was adjusted to near neutral with saturated barium hydroxide aqueous solution, and dried under reduced pressure. Water was added to the obtained concentrate again, and it dried under reduced pressure again. This operation was repeated once more. A small amount of water was added to the obtained concentrate to remove the precipitate of barium sulfate by centrifugation, and the obtained supernatant was used for a cation exchange column [Amberlite IRA-120 (Na ⁺ ) (Organo)]. The resulting column fraction was concentrated under reduced pressure to prepare 700 mg of sulfated D-(+)-glucose sodium salt.
[216] (2) 240 mg (1.3 mmol) of D-(+)-galactose was dissolved in 10 ml of pyridine, 1.05 g (6.6 mmol) of Pyr.SO ₃ was added at room temperature, followed by water at room temperature and 60 ° C for 1 hour. By stirring, 406 mg of sulfated D-(+)-galactose sodium salt was prepared in the same manner as in Reference Example 14- (1) below.
[217] (3) 200 mg (1.3 mmol) of D-(+)-mannose was dissolved in 10 ml of pyridine, 1.05 g (6.6 mmol) of Pyr.SO ₃ was added at room temperature, followed by water at room temperature and 60 ° C for 1 hour. By stirring, 700 mg of sulfated D-(+)-mannose sodium salt was prepared in the same manner as in Reference Example 14- (1) below.
[218] (4) 205 mg (0.57 mmol) of maltose were dissolved in 10 ml of pyridine, 816 mg (5.2 mmol) of Pyr.SO ₃ was added at room temperature, followed by stirring at room temperature for 1 hour at 60 ° C. In the same manner as in 14- (1), 520 mg of sulfated maltose sodium salt was prepared.
[219] (5) 200 mg (0.4 mmol) of maltotriose were dissolved in 10 ml of pyridine, 700 mg (4.4 mmol) of Pyr.SO ₃ was added at room temperature, followed by stirring at room temperature for 1 hour at 60 ° C, and then In the same manner as in Reference Example 14- (1), 420 mg of sulfated maltotriose sodium salt was prepared.
[220] (6) 250 mg (0.73 mmol) of trehalose were dissolved in 10 ml of pyridine, 1.1 g (7 mmol) of Pyr.SO ₃ was added at room temperature, followed by stirring at room temperature for 1 hour at 60 ° C, 750 mg of sulfated trehalose sodium salt was prepared in the same manner as in Reference Example 14- (1) below.
[221] (7) 222 mg (0.62 mmol) of lactose were dissolved in 10 ml of pyridine, 785 mg (4.9 mmol) of Pyr.SO ₃ was added at room temperature, followed by stirring at room temperature for 1 hour at 60 ° C. 476 mg of sulfated lactose sodium salt was prepared by the same operation as 14- (1).
[222] (8) 220 mg (0.62 mmol) of sucrose was dissolved in 10 ml of pyridine, and 785 mg (4.9 mmol) of Pyr.SO ₃ was added at room temperature, followed by stirring at room temperature for 1 hour at 60 ° C. 481 mg of sulfated sucrose sodium salt was prepared by the same operation as 14- (1).
[223] (9) 370 mg (1.08 mmol) of lactulose were dissolved in 10 mL of pyridine, 1.38 g (8.8 mmol) of Pyr.SO ₃ was added at room temperature, followed by stirring at room temperature for 1 hour at 60 ° C, followed by stirring for 1 hour. 1 g of sulfated lactulose sodium salt was prepared in the same manner as in Reference Example 14- (1).
[224] (10) 379 mg (0.9 mmol) of melibiose was dissolved in 10 ml of pyridine, and 1.43 mg (9.0 mmol) of Pyr.SO ₃ was added at room temperature, followed by stirring at room temperature for 1 hour at 60 ° C, followed by reference. 950 mg of sulfated melibiose sodium salt was prepared in the same manner as in Example 14- (1).
[225] (11) 150 mg (1.0 mmol) of D-(+)-xylose was dissolved in 10 ml of pyridine, and 770 mg (4.8 mmol) of Pyr.SO ₃ was added at room temperature, followed by water at room temperature and 60 ° C. After stirring for 1 hour, 350 mg of sulfated D-(+)-xylose sodium salt was prepared in the same manner as in Reference Example 14- (1) below.
[226] (12) 200 mg (1.2 mmol) of 2-deoxy-glucose was dissolved in 10 ml of pyridine, 920 mg (5.8 mmol) of Pyr.SO ₃ was added at room temperature, followed by stirring at room temperature for 1 hour at 60 ° C. In the same manner as in Reference Example 14- (1) below, 500 mg of sulfated 2-deoxy-glucose sodium salt was prepared.
[227] (13) 150 mg (0.83 mmol) of D-glucitol was dissolved in 10 ml of pyridine, 955 mg (6 mmol) of Pyr.SO ₃ was added at room temperature, followed by stirring at room temperature for 1 hour at 60 ° C. , 570 mg of sulfated D-glucitol sodium salt was prepared in the same manner as in Reference Example 14- (1) below.
[228] (14) 147 mg (0.43 mmol) of cellobiose were dissolved in 5 ml of dimethyl sulfoxide, 657 mg (4.13 mmol) of Pyr.SO ₃ was added at room temperature, followed by stirring at room temperature for 1 hour at 60 ° C, In the same manner as in Reference Example 14- (1) below, 230 mg of sulfated cellobiose sodium salt was obtained.
[229] (15) 62 mg (0.18 mmol) of isomaltose were dissolved in 5 ml of pyridyl, 275 mg (1.73 mmol) of Pyr.SO ₃ was added at room temperature, followed by stirring at room temperature for 1 hour at 60 ° C, followed by stirring for 1 hour. 162 mg of sulfated isomaltose sodium salt was obtained by the same operation as the Reference Example 14- (1).
[230] (16) 293 mg (0.86 mmol) of turanose was dissolved in 5 ml of pyridyl, 1310 mg (8.22 mmol) of Pyr.SO ₃ was added at room temperature, followed by stirring at room temperature for 1 hour at 60 ° C, Hereinafter, 835 mg of sulfated turanose sodium salt was obtained by the same operation as the Reference Example 14- (1).
[231] (17) 315 mg (0.875 mmol) of palatinose were dissolved in 5 ml of pyridyl, 1.34 mg (8.4 mmol) of Pyr.SO ₃ was added at room temperature, followed by stirring at room temperature for 1 hour at 60 ° C, followed by stirring for 1 hour. 845 mg of sulfated palatinose sodium salt was obtained in the same manner as in Reference Example 14- (1).
[232] (18) 56 mg (0.31 mmol) of α-D-talose were dissolved in 5 ml of pyridyl, and 300 mg (1.9 mmol) of Pyr.SO ₃ was added at room temperature, followed by water at room temperature and 60 ° C. for 1 hour. It stirred, and 150 mg of sulfated D- thalose sodium salts were obtained by operation similar to the following reference example 14- (1).
[233] (19) A total acetylated product of maltohexaose was obtained by treating a fully acetylated product of 7 g of α-cyclodextrin with a mixed solution of acetic anhydride and sulfuric acid (49: 1), which was converted into sodium methoxide (NaOMe) in methanol. Deacetylation gave 1.5 g of maltohexaose. 79 mg (0.83 mmol) of maltohexaose and 1.33 g of piperidine sulfate were dissolved in 5 ml of dimethyl sulfoxide (DMSO), followed by stirring at 80 ° C. for 2 hours. The reaction solution was cooled and then dialyzed for 2 days with a dialysis membrane having a molecular weight of 1000 cuts. The dialysis solution obtained was used for a cation exchange column [Amberlite IRA-120 (Na ⁺ ) (Organo)]. The column pass fraction thus obtained was concentrated under reduced pressure to prepare 167 mg of sulfated maltohexaose sodium salt.
[234] (20) 2.2 g of β-cyclodextrin was treated with a mixture of acetic anhydride and sulfuric acid (49: 1) to obtain a completely acetylated product of maltoheptaose, which was deacetylated with NaOMe in methanol to treat maltohepta. 0.5 g of os was obtained. 20 mg (0.83 mmol) of maltoheptaose and 325 mg of piperidine sulfate were dissolved in 5 ml of DMSO and stirred at 80 ° C. for 2 hours, followed by sodium sulfate of maltoheptaose in the same manner as in Reference Example 14- (19) below. 45.6 mg of salt were prepared.
[235] (21) The acetylated maltohexaose was obtained by stirring the complete acetylated product of maltohexaose in dichloromethane in the presence of trichloroacenitrile and potassium carbonate. Imidate of acetylated maltohexaose and dodecanol were reacted with trifluoromethanesulfonic acid trimethylsilyl in dichloromethane as a catalyst, and dodecyl-maltohexaose was obtained by deacetylating the obtained reactant. 370 mg (0.32 mmol) of dodecyl-maltohexaose were dissolved in 10 ml of DMSO and stirred at 80 ° C. for 2 hours, followed by sulfated dodecyl-maltohexaose sodium salt in the same manner as in Reference Example 14- (19) below. 700 mg was prepared.
[236] (22) 276 mg of starch was dissolved in 10 ml of DMSO, and 2.76 g of Pyr.SO ₃ was added at room temperature, followed by stirring at 80 ° C for 2 hours. After cooling the reaction solution, the insoluble fraction formed by adding acetone was washed several times with methanol, and then diluted with water and used for a cation exchange column [Amberlite IRA-120 (Na ⁺ ) (Organo)]. The column pass fraction thus obtained was concentrated under reduced pressure to prepare 350 mg of sulfated starch sodium salt.
[237] (23) Cardane 111 mg was dissolved in 5 ml of DMSO, and 1.11 g of Pyr.SO ₃ was added at room temperature, followed by stirring at 80 ° C for 2 hours. After cooling the reaction solution, acetone was added, the resulting insoluble fraction was diluted with water, neutralized to near pH neutral with saturated sodium bicarbonate water, and dialyzed with a dialysis membrane having a molecular weight of 1000 cuts for 1 day. The obtained dialysate solution was supplied to a cation exchange column [Amberlite IRA-120 (Na ⁺ ) (Organo)], and then dried under reduced pressure to prepare 180 mg of sodium sulfate cadmium salt.
[238] (24) 267 mg of pectin was dissolved in 5 ml of DMSO, and 2.67 g of Pyr.SO ₃ was added at room temperature, followed by stirring at 80 ° C for 2 hours. After cooling the reaction solution, 384 mg of sulfated pectin sodium salt was prepared in the same manner as in Reference Example 14- (23).
[239] Example 1
[240] (1) 500 μl of MRC-5 cells (CCL171: Dainippon Seiyakusha, code. 02-021) suspended in DME medium containing 10% fetal bovine serum to 1 × 10 ⁵ cells / mL was used for 48 well cells. The plates were placed in culture plates and exchanged with DME medium containing 1% fetal bovine serum after 24 hours of incubation at 37 ° C. in the presence of 5% CO ₂ . Thereafter, Kagome kelp-derived fucoidan described in Reference Example 1- (1) was added as a sample so as to have a final concentration of 1, 10, 100 µg / ml, and cultured again for 24 hours, and then the medium was recovered. Growth Factor (HGF) The amount of HGF in the medium was measured using an ELISA Kit (manufactured by Funakoshisha, Code.RS-0641-00).
[241] As a control, the same amount of distilled water as the sample was added. The HGF amount of the control group is 7.2 ng / ml, and the HGF production amount of each sample addition group having this value as 100% is shown in Table 1. In addition, all the experiments were performed twice continuously and the average value was employ | adopted.
[242] Kagome kelp-derived fucoidan (µg / ml)Production of HGF (%) 0100 One214 10339 100339
[243] In the Kagome kelp-derived fucoidan-added group, the HGF production increased significantly compared to the distilled water-added control group. In addition, the production of HGF significantly increased compared to the case of adding heparin or low molecular weight heparin, so that the Kafume kelp-derived fucoidan has a higher HGF than heparin, which has been confirmed to produce HGF, or a low molecular weight heparin with an average molecular weight of about 5000. It has been shown to have activity to promote production.
[244] (2) 7-12SFd-F prepared in the same manner as in Example 1- (1), prepared by the method described in Reference Example 1- (2), II fraction, III fraction, or Reference Example 2; HGF production induction of each of 6-2S prepared by the method described in Reference Example 4, seaweed algae-derived fucoidan prepared by the method described in Reference Example 6, and hatban-derived fucoidan prepared by the method described in Reference Example 7 was measured. The results are shown in Tables 2-4.
[245] sampleConcentration (µg / ml)Production of HGF (%) Ⅰ fractionOne167 100234 Ⅱ fractionOne208 100359 Ⅲ fractionOne146 100291
[246] (HGF production in the control was 8.3 ng / ml)
[247] sampleConcentration (µg / ml)Production of HGF (%) Fucoidan from HatbanOne148 10246 100335 Fucoidan originated from seaweedOne179 10250 100291 7-12SFd-FOne149 10276 100339
[248] (HGF production of the control was 8.6 ng / ml)
[249] sampleConcentration (µg / ml)Production of HGF (%) 6-2S10112 100246
[250] (HGF production of the control was 9.9 ng / ml)
[251] Induction of strong HGF production from fractions of Kagome kelp-derived fucoidans, namely U-fucoidan, F-fucoidan, hatban-derived fucoidan, seaweed-derived fucoidan, F-fucoidan-derived 7-12SFd-F and U-fucoidan-derived 6-2S, respectively This was recognized. In addition, the kelp-derived fucoidan described in Reference Example 1- (1), the fucoidan derived from resonia nigressen, the ascofilum nodo island-derived fucoidan described in Reference Example 7, the acid decomposed product described in Reference Example 8, and the A to F fractions were also strong. Induction of HGF production was recognized.
[252] (3) -① A 2% solution of Kagome kelp-derived fucoidan prepared by the method described in Reference Example 1- (1) was prepared with citric acid or sulfuric acid at pH 3, each was heated to 100 ° C, and after 30 minutes, 1 After 2 hours, after 4 hours, each hydrolyzate was prepared, and the HGF production induction was measured under the same conditions as in 1- (1). Was used.
[253] sampleHeating timeProduction of HGF (%) Undigested liquid 448 Sulfuric Acid30 minutes364 1 hours385 2 hours368 4 hours345 Undigested liquid 480 Citric Acid Degradation Solution30 minutes402 1 hours429 2 hours397 4 hours341
[254] (HGF production of the control was 8.6 ng / ml)
[255] (3) -② The heat treated product in the presence of citric acid of Kagome kelp-derived fucoidan prepared in Example 1- (3) -① was fractionated by gel filtration.
[256] That is, a column packed with 1.5 liters of Toyo Pearl HW40C was equilibrated with water, and 10 ml of a heat treatment of Kagome kelp-derived fucoidan was applied to the column, and then eluted with water at a flow rate of 1 ml / min. The first 680 mL was eluted as it was, and after that, it fractionated every 14 mL, and the gel filtration fraction of the heat process was obtained.
[257] This fraction was analyzed by TLC (solvent, butyl acetate: acetic acid: water = 3: 4: 3, detection agent osionol sulfuric acid), and according to the pattern of dots, fractions 12 to 13, 16 to 17, 26 to 40, and the like. The gel filtration fractions were combined and lyophilized. The lyophilisate of each fraction obtained was redissolved in water so as to be 100 mg / ml, and the action of HGF production induction was measured under the same conditions as in Example 1- (1).
[258] As a result, HGF production-inducing activity was recognized in each of the fractions 12-13 and 16-17.
[259] Fractions of the fractions 12 to 13 were subjected to structural determination, and the analytical values thereof were consistent with the analytical values of the compounds represented by the following formula VII in pamphlet No. 97/26896. Production induction activity was recognized.
[260]
[261] (4) A commercially available solution of sodium dextran sulfate (manufactured by Sigma) was prepared, and the action of inducing HGF production was measured according to the method described in Example 1- (1). As shown in Table 6, sodium dextran sulfate exhibited HGF production induction.
[262] Dextran sodium sulfate (average molecular weight)Concentration (µg / ml)Production of HGF (%) 1 millionOne588 10655 100787 8 thousandOne395 10573 100695 Five thousandOne398 10421 100565
[263] (HGF production of the control was 6.7 ng / ml)
[264] (5) A solution of a commercially available -Carrageenan (manufactured by Nakarai Tessha) was prepared, and the action of HGF production induction was measured according to the method of Example 1- (1). As shown in Table 7, λ-carrageenan exhibited HGF production induction.
[265] λ-carrageenan (μg / ml)Production of HGF (%) One152 10140
[266] (HGF production of the control was 13.4 ng / ml)
[267] (6) -① A commercially available alginic acid solution (manufactured by Wako Pure Chemical Industries, Ltd., swellable) was prepared, and the action of HGF production induction was measured according to the method of Example 1- (1). As shown in Table 8, alginic acid exhibited HGH production induction.
[268] Alginic acid (µg / ml)Production of HGF (%) One125 10154 100327
[269] (The HGF amount in the control group was 7.3 ng / ml)
[270] (6) -② Similarly, alginic acid (swelling, manufactured by Wakojun Yakusha: sample ①), alginic acid (non-swellable, manufactured by Wakojun Yakusha: sample ②), alginic acid (100-150 cp, manufactured by Wakojunya Kusha: sample ③), The HGF production-inducing activity of alginic acid (300-400 cp, manufactured by Wakojunya Co., Ltd .: Sample ④) and alginic acid (500-600 cp, manufactured by Wako Junya Co .: Sample ⑤) was examined. As shown in Table 9, all of the samples ① and ⑤ induced the production of HGF. As mentioned above, it became clear that alginic acid which is an acidic polysaccharide also has HGF production induction activity.
[271] Concentration (µg / ml)Production of HGF (%) Sample ①Sample ②Sample ③Sample ④Sample ⑤ 10154138115127104 100327158184152187
[272] (HGF production was 7.3 ng / ml for the controls ① and ② and 6.7 ng / ml for the controls ③, ④ and ⑤).
[273] (6) -③ Similarly, the HGF production | induction activity of pectinic acid (made by Nakarai Tessha) was examined. As shown in Table 10, pectinic acid induced the production of HGF.
[274] As mentioned above, it became clear that the acid polysaccharide, pectinic acid, also has HGF production inducing activity.
[275] sampleConcentration (µg / ml)Production of HGF (%) Factinic acidOne145 10218 100684
[276] (HGF production of the control was 5.91 ng / ml)
[277] (7) The HGF production inducing activity of salmon sperm DNA (Nichirosha Co., Ltd.) was examined. DNA was added so that the final concentration was 1, 10, 100 µg / ml. As shown in Table 11, salmon sperm DNA showed HGF production-inducing activity.
[278] sampleConcentration (µg / ml)Production of HGF (%) Salmon Sperm DNAOne110 10182 100285
[279] (HGF production of the control was 9.9 ng / ml)
[280] (8) A solution of the large horse-derived fucoidan and sea cucumber-derived fucoidan prepared in Reference Examples 9 and 11 was prepared, and the HGF production induction action was measured according to the method of Example 1- (1). As shown in Table 12, each fucoidan exhibited HGF production induction.
[281] sampleConcentration (µg / ml)Production of HGF (%) Fucoidan from sea cucumberOne282 10356 100495 Fucoidan from big horseOne218 10279 100307
[282] (The HGF amount of the control group was 7.77 ng / ml)
[283] (9) Prepared by preparing a solution of sulphated polysaccharide (sample ①), sulfated polysaccharide (sample ②) and heterocradiated polysulphate (sample ③) prepared in Reference Example 10. HGF production-inducing activity was examined in the same manner as in Example 1- (1). Samples ① and ③ were added so as to have a final concentration of 1, 10 and 100 µg / ml, and sample ② was added so as to have a final concentration of 10 and 100 µg / ml. As shown in Table 13, samples ① and ③ all induced the production of HGF.
[284] sampleConcentration (µg / ml)Production of HGF (%) Sample ①One100 10177 100169 Sample ②10117 100198 Sample ③One116 10207 100228
[285] (HGF production of the control was 7.3 ng / ml)
[286] (10) In the same manner as in Example 1- (1), the Resonia-derived fucoidan prepared in Reference Example 10- (3) (sample ①), DEAE33 fraction (sample ②), DEAE37 fraction (sample ③), and DEAE40 fraction ( The HGF production-inducing activity of sample ④) was examined. Each sample was added to a final concentration of 1, 10, 100 μg / ml. As shown in Table 14, all of the samples ④ in the sample ① induced the production of HGF.
[287] Concentration (µg / ml)Production of HGF (%) Sample ①Sample ②Sample ③Sample ④ One138105235121 10167221253254 100331265261295
[288] (HGF production of the control was 11.5 ng / ml)
[289] (11) Sulfated fucogalactan as described in Reference Example 3- (2), agalopectin as described in Reference Example 12, chondroitin sulfate B (manufactured by Sekagaku Kogyosha), and chondroitin under the same conditions as in Example 1- (1). The HGF production-inducing activity of sulfuric acid D (made by Sekagaku Kogyosha) was examined. Each sample was added to a final concentration of 1, 10, 100 μg / ml. As shown in Tables 15-17, sulfated fucogalactan, agalopectin and chondroitin sulfate induced the production of HGF.
[290] sampleConcentration (µg / ml)Production of HGF (%) Sulfated FucogalactanOne194 10355 100429
[291] (HGF production of control was 4.3 ng / ml)
[292] sampleConcentration (µg / ml)Production of HGF (%) AgaropectinOne117 10121 100256
[293] (HGF production of the control was 6.7 ng / ml)
[294] sampleConcentration (µg / ml)Production of HGF (%) Chondroitin Sulfate BOne117 10121 100256 Chondroitin Sulfate D10119 100144
[295] (HGF production of control was 11.9 ng / ml)
[296] (12) Spirulina derived from Reference Examples 13- (1), 13- (3), 13- (5), 13- (7) and 13- (8) in the same manner as in Example 1- (1), respectively. HGF production-inducing activities of polysulfated sulfate, polychlorinated sulfate-derived polysaccharides, polysaccharides from mugwort, polysaccharides from vines, polysaccharides derived from aloe vera, polysaccharides derived from aloe foliar, and sulfated polysaccharides derived from aloe leaf surface were examined. The polysaccharide and the mugwort-derived polysaccharide were added such that their final concentration was 1, 10, 100 µg / ml. Chlorella-derived polysaccharides, vine liposide-sulfated polysaccharides, aloe foliar-derived polysaccharides, and aloe leaf surface-derived polysaccharides were added such that their final concentrations were 1, 10, 100, and 1000 μg / ml. As shown in Tables 18 to 20, spirulina-derived polysaccharides, chlorella-derived polysaccharides, mugwort-derived polysaccharides, vine lipopolysaccharide-sulfated polysaccharides, aloe leaf meat-derived polysaccharides, and aloe leaf surface-derived polysaccharides The production of HGF was induced.
[297] sampleConcentration (µg / ml)Production of HGF (%) Spirulina-derived polysaccharide sulfateOne149 10293 100398 Chlorella-derived polysulfateOne108 10149 100175 1000396
[298] (HGF production of the control was 7.9 ng / ml)
[299] sampleConcentration (µg / ml)Production of HGF (%) Sulfate-derived polysaccharideOne137 10284 100265
[300] (HGF production of the control was 12.7 ng / ml)
[301] sampleConcentration (µg / ml)Production of HGF (%) Sulfated polysaccharides derived from vinesOne111 10104 100133 1000190 Polysulfuric acid derived from aloe leaf meatOne111 10125 100150 1000401 Polysaccharide Sulfate Derived from Aloe Leaf SurfaceOne106 10125 100120 1000328
[302] (HGF production of the control was 8.7 ng / ml)
[303] (13) In the same manner as in Example 1- (1), the spirulina fraction SSP-I (sample ①), SSP-II (sample ②), SSP-III (sample ③), prepared in Reference Example 13- (2), The HGF production-inducing activity of SSP-IV (sample ④) was examined. Each sample was added to a final concentration of 1, 10, 100 μg / ml. As shown in Table 21, all of the samples ① and ④ induced the production of HGF.
[304] Concentration (µg / ml)Production of HGF (%) Sample ①Sample ②Sample ③Sample ④ One240165141218 10243152270282 100302212280351
[305] (Control HGF production was 7.3 ng / ml)
[306] (14) HGF production-inducing activity of the CSP-I fraction (sample ①) and CSP-II fraction (sample ②) of the chlorella extract prepared in Reference Example 13- (4) in the same manner as in Example 1- (1). Reviewed. Sample ① was added at a final concentration of 10, 100 µg / ml, and sample ② at a final concentration of 100 µg / ml. As shown in Table 22, samples ① and ② induced the production of HGF.
[307] sampleConcentration (µg / ml)Production of HGF (%) Sample ①10111 100173 Sample ②100175
[308] (HGF production of the control was 11.4 ng / ml)
[309] (15) YAP fraction (Sample ①), YSP-I fraction (Sample ②), YSP-II fraction (Sample ③) of mugwort extract prepared in Reference Example 13- (6) in the same manner as in Example 1- (1) ), YSP-II-2 fraction (Sample ④), YSP-II-3 fraction (Sample ⑤), and YSP-II-4 fraction (Sample ⑥) were investigated. Each sample was added to a final concentration of 1, 10, 100 μg / ml. As shown in Table 23 and Table 24, all of the samples ⑥ in the sample ① induced the production of HGF. In particular, strong HGF production-inducing activity was observed in the YSP-II fraction (sample ③), the YSP-II-3 fraction (sample ④), and the YSP-II-4 fraction (sample ⑤).
[310] sampleConcentration (µg / ml)Production of HGF (%) Sample ①10121 100266 Sample ②One104 10148 100381 Sample ③One328 10386 100390
[311] (HGF production of the control was 11.5 ng / ml)
[312] Concentration (µg / ml)Production of HGF (%) Sample ④Sample ⑤Sample ⑥ One152290226 10462383322 100475321314
[313] (HGF production of the control was 7.7 ng / ml)
[314] (16) Sulfated maltose sodium salt, sulfated maltotriose sodium salt, sulfated lactose sodium salt, sulfated sucrose sodium salt and sulfated trehalo prepared in Reference Example 14 in the same manner as in Example 1- (1) Os sodium salt, sulfated glucose sodium salt, sulfated lactose sodium salt, sulfated melibiose sodium salt, sulfated galactose sodium salt, sulfated mannose sodium salt, sulfated xylose sodium salt, sulfated 2-dioxy -Glucose Sodium Salt, Sulfated Glucitol Sodium Salt, Sulfated Cellobiose Sodium Salt, Sulfated Isomaltose Sodium Salt, Sulfated Turanose Sodium Salt, Sulfated Palatinose Sodium Salt, Sulfated Talos Sodium Salt, Sulfated maltohexaose sodium salt, sulfated maltoheptaose sodium salt, sulfated dodecyl-maltohexaose sodium salt, sulfated starch sodium salt, sulfated crananthate Salts, sulfated pectin sodium salt HGF production inducing activity was examined. Each sample was added to a final concentration of 1, 10, 100 μg / ml, or 10, 100, 1000 μg / ml, or 100 μg / ml. As a control, the same amount of distilled water as the sample was added. In addition, each sugar that was not sulfated was measured to induce HGF production at the same concentration as each sulfated sugar.
[315] As shown in Tables 25-34, sulfated oligosaccharides and sulfated monosaccharides induced the production of HGF. In addition, each sugar that was not sulfated did not induce HGF.
[316] From the results of the sulfated dodecyl-maltohexaose sodium salt, it became clear that HGF production-inducing activity was maintained even when the sugar was modified by lipids.
[317] sampleConcentration (µg / ml)Production of HGF (%) Sulfated Maltose Sodium SaltOne142 10273 100439 Sulfated Maltotriose Sodium SaltOne151 10286 100387
[318] (HGF production of the control was 8.7 ng / ml)
[319] sampleConcentration (µg / ml)Production of HGF (%) Sulfated Lactose Sodium SaltOne118 10185 100410 Sulfated Sucrose Sodium SaltOne173 10355 100501 Sulfated Glucose Sodium SaltOne178 10377 100864
[320] (HGF production of control was 5.5 ng / ml)
[321] sampleConcentration (µg / ml)Production of HGF (%) Sulfated Trehalose Sodium SaltOne277 10447 100421
[322] (The production of control HGF was 4.3 ng / ml)
[323] sampleConcentration (µg / ml)Production of HGF (%) Sulfated Galactose Sodium Salt100166
[324] (HGF production of the control was 12.7 ng / ml)
[325] sampleConcentration (µg / ml)Production of HGF (%) Sulfated Mannose Sodium SaltOne112 10175 100456
[326] (HGF production of the control was 6.2 ng / ml)
[327] sampleConcentration (µg / ml)HGF Production (%) Sulfated Lactose Sodium Salt10139 100376 1000583 Sulfated melibiose sodium salt10240 100403 1000667 Sulfated Xylose Sodium Salt10110 100112 1000284 Sulfated 2-Dioxyglucose Narlium Salt10127 100102 1000239 Sulfated glutitol sodium salt10112 100203 1000335
[328] (HGF production of the control was 5.9 ng / ml)
[329] sampleConcentration (µg / ml)HGF Production (%) Sulfated Cellobiose Sodium Salt10100 100143 1000546 Sulfated Isomaltose Sodium Salt10134 100301 1000477 Sulfated Turanose Sodium Salt10127 100203 1000379 Sulfated Pallatinose Narrium Salt10132 100278 1000519
[330] (HGF production in the control group was 11.1 ng / ml of sulfated cellobiose sodium salt, 11.3 ng / ml of sulfated isomaltose sodium salt, 8.6 ng / ml of sulfated turanose sodium and sulfated pallatinose sodium salt)
[331] sampleConcentration (µg / ml)Production of HGF (%) Sulfated Tallowose Sodium Salt10112 100263 1000494
[332] (HGF production of the control was 9.5 ng / ml)
[333] sampleConcentration (µg / ml)Production of HGF (%) Sulfated Maltohexaose Sodium Salt10407 100571 1000761 Sulfated Maltoheptaose Sodium Salt10341 100486 1000706 Sulfated Dodecyl-Maltohexaose Sodium Salt10289 100371 1000359
[334] (HGF production of the control was 8.15 ng / ml)
[335] sampleConcentration (µg / ml)Production of HGF (%) Sulfated starch sodium salt10781 100864 1000804 Sulfated Guardan Sodium Salt10359 100503 1000617 Sulfated Pactin Sodium Salt10721 100780 1000648
[336] (HGF production of the control was 8.15 ng / ml)
[337] Example 2
[338] (1) In the same manner as in Example 1- (1), the synergistic effect on the induction of HGF production between Kagome Kelp-derived fucoidan, prostaglandin and IL-1 described in Reference Example 1- (1) was examined.
[339] That is, the synergistic effect of HGF production inducing activity was examined by simultaneously adding the fucoidan, PGE ₁ (manufactured by Wako Pure Chemical Industries, Ltd.), and IL-1α (manufactured by Genzyme Co.).
[340] Fucoidan samples were added so that the final concentration was 1, 10, 100 µg / ml. PGE ₁ was added to 0.1, 1, μM, and IL-1α to 1 ng / ml. As a control, the same amount of distilled water as the sample was added.
[341] The synergistic effect of the fucoidan sample or PGE ₁ and IL-1α alone was compared with each other, and the synergistic effect was examined.
[342] The results are shown in Tables 35 and 36. In Tables 35 and 36, the HGF production amount of the control group was shown as 100%. All experiments were carried out twice in succession and the average value was adopted. As shown in Tables 35 and 36, synergistic effects on the induction of HGF were recognized by simultaneous addition of fucoidan and PGE ₁ or IL-1α.
[343] Fucoidan addition amount (µg / mL)PGE ₁ addition amount (μM) 00.1One Production of HGF (%) 0100119157 One195334415 10331517561 100382731682
[344] Fucoidan addition amount (µg / mL)IL-1α addition amount (ng / ml) 0One Production of HGF (%) 0100163 One206421 10350672 100403780
[345] (HGF production of the control was 9.1 ng / ml)
[346] (2) The synergistic effect on the induction of HGF production between 7-12SFd-F, prostaglandin and IL-1 in Reference Example 2 was examined in the same manner as in Example 2- (1). The synergistic effect of HGF production-inducing activity was examined by adding 7-12SFd-F, PGE ₁ , and IL-1α simultaneously. Each 7-12SFd-F was added to a final concentration of 1, 10, 100 μg / ml. To each 7-12SFd-F-added cells, PGE ₁ and IL-1α were additionally added at the same time. PGE ₁ was added to 0.1, 1 μM, and IL-1α to 0.1, 1 ng / ml. As a negative control, the same amount of distilled water as the sample was added. The synergistic effect was examined in comparison with the production amount of 7-12SFd-F or PGE ₁ and IL-1α alone, respectively. The production amount of HGF was represented as 100% of the negative control in which only distilled water was added. The results are shown in Tables 37 and 38. All experiments were performed three times in succession, and the average value was adopted.
[347] 7-12SFd-F addition amount (㎍ / mL)PGE ₁ addition amount (μM) 00.1One Production of HGF (%) 0100123170 One120158216 10218309317 100219365382
[348] (HGF production in the control was 5.1 ng / ml)
[349] 7-12SFd-F addition amount (㎍ / mL)IL-1α addition amount (ng / ml) 00.1One Production of HGF (%) 0100130151 One140168159 10191154208 100203255222
[350] (HGF production in the control was 5.1 ng / ml)
[351] Example 3
[352] (1) 48 well cells containing 500 μl of KG-1-C cells (Glomer: Human Science Shinkozaidan) suspended in DMEM medium containing 10% fetal bovine serum to 1 × 10 ⁵ cells / ml. The plate was placed in a culture plate and exchanged with DMEM medium containing 1% fetal bovine serum after overnight incubation at 37 ° C. in the presence of 5% CO ₂ . Then, after adding the test sample and incubating for another 20 hours, the medium was recovered and the amount of HGF in the medium was measured using the HGF ELISA kit described in Example 1.
[353] The test sample was added so that the final concentration was 1, 10, 100 µg / ml for Kagome Kelp-derived fucoidan described in Reference Example 1- (1), and 1, 10 µg / ml for Heparin (manufactured by Wako Pure Chemical Industries, Ltd.). It was. As a control, the same amount of distilled water as the sample was added. All experiments were performed three times in succession, and the average value was adopted. The results are shown in Table 39. In Table 39, the HGF production amount of the control group was shown as 100%.
[354] All of the cell groups to which the fucoidan sample was added had significantly increased HGF production than the control group to which distilled water was added. In addition, the production of HGF was significantly higher than that of heparin. As a result, the fucoidan was found to have a higher HGF production activity than heparin, which has been confirmed to induce HGF.
[355] sampleConcentration (µg / ml)Production of HGF (%) Fucoidan from Kagome KelpOne194 10285 100351 Heparin Sodium SaltOne176 10215
[356] (HGF production of the control was 4.4 ng / ml)
[357] (2) HL-60 cells (promyelocytic leukemia cells: ATCC CCL-240) cultured in RPMI1640 medium containing 10% fetal bovine serum were added to RPMI1640 medium containing 1% fetal bovine serum to 1 x 10 ⁵ cells / ml. Suspension was placed in 48 well cell culture plates at 500 μl. Thereafter, 10 nM of 12-O tetradecanoyl phorbol 13-acetate (TPA: manufactured by GIBCO BRL) was added, and a test sample was further added simultaneously. After incubation for 20 hours, the medium was recovered, and the amount of HGF in the medium was measured using an HGF ELISA kit.
[358] The test sample was added such that the final concentration of Kagome Kelp-derived fucoidan described in Reference Example 1- (1) was 1, 10, and 100 µg / ml. Heparin was added to 1, 10 µg / ml. As a control, the same amount of distilled water as the sample was added. All experiments were performed three times in succession, and the average value was adopted. The results are shown in Table 40. In Table 40, the production amount of HGF of the control group was represented as 100%. All of the cell groups to which the fucoidan sample was added had significantly increased HGF production than the control group to which distilled water was added. In addition, the production of HGF was significantly higher than that of heparin. As a result, the fucoidan was found to have a higher HGF production activity than heparin, which has been confirmed to induce HGF.
[359] sampleConcentration (µg / ml)Production of HGF (%) Fucoidan from Kagome KelpOne191 10342 100490 Heparin Sodium SaltOne140 10189
[360] (The production amount of HGF of the control group was 0.4 ng / ml).
[361] Example 4
[362] 1 × 10 into the MRC-5 sepoaek 500 ㎕ suspended in DME medium containing 10% fetal bovine serum so that the ⁵ cells / ㎖ a cell culture plate with 48 wells, 37 ℃, after 24 hours incubation under 5% CO ₂ present Exchange with DME medium containing 1% fetal bovine serum. Thereafter, the sample was added and incubated again for 24 hours. This medium was recovered and the amount of HGF in the medium was measured using an HGF ELISA kit. In addition, after washing the cells with PBS, 500 μl of cytolysis buffer (50 mM HEPES pH 7.4, 10 mM EDTA, 0.1% Triton × 100, 1 mM PMSF, 1 μg / ml pepstatin A, 1 μg / ml leupetin )). After sonication for more complete lysis, the supernatant (cell extract) was prepared by centrifugation, and the amount of HGF in the cells was measured to be equal to the concentration of HGF in the medium.
[363] Kagome kelp-derived fucoidan described in Reference Example 1- (1) as a test sample was added so as to have a final concentration of 1, 10, 100 µg / ml. As a control, the same amount of distilled water as the sample was added. All experiments were carried out twice in succession and the average value was adopted. The results are shown in Table 41. As shown in Table 41, the amount of HGF in the medium of the corresponding fucoidan addition group increased significantly in the concentration of fucoidan compared to the control group of the distilled water addition. On the other hand, the amount of HGF in the cells decreased Fucoidan concentration dependent. Next, the total HGF amount inside and outside the cells increased in a concentration-dependent manner. As a result, the fucoidan was found to have an activity of promoting the production of HGF and an action of promoting the release of HGF from cells.
[364] Kagome kelp-derived fucoidan (µg / ml)Production of HGF (ng / well) BadgeIn the cellWhole quantity Control4.25.79.8 One9.33.212.5 1015.90.916.8 10016.90.417.3
[365] Example 5
[366] (1) 500 μl of MRC-5 cell solution suspended in DME medium containing 10% fetal bovine serum to 1 × 10 ⁵ cells / ml is placed in a 48 well cell culture plate, and is placed at 37 ° C. in the presence of 5% CO _2. After time incubation it was exchanged with DME medium containing 1% fetal bovine serum. Thereafter, a sample was added and cultured again for 0, 0.5, 1, 2, 4, 8, 12, 24 hours, and then the medium was recovered, and the amount of HGF in the medium was measured using an HGF ELISA kit. Kagome kelp-derived fucoidan described in Reference Example 1- (1) was added so as to have a final concentration of 10 µg / ml. As a control, the same amount of distilled water as the sample was added. The results are shown in Table 42. As shown in Table 42, the fucoidan-added group significantly increased HGF production time-dependently than the control group.
[367] As a result, the fucoidan has a high HGF production promoting activity, the HGF production was found to increase over time.
[368] Incubation time (hours) 00.5One2481224 Production of HGF (ng / mL) Control0.020.030.810.902.313.906.949.39 Fucoidan addition0.191.905.756.879.0415.920.131.3
[369] (2) 500 µl of MRC-5 cells (CCL 171: Dainippon Seiyaku, code. 02-021) suspended in DME medium containing 10% fetal bovine serum to 1 x 10 ⁵ cells / ml was prepared in 48 wells. Cell culture plates were placed and exchanged with DME medium containing 1% fetal bovine serum after 24 hours of incubation at 37 ° C. in the presence of 5% CO ₂ . Thereafter, the sample was added, and cultured again after 0, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours, and the medium was recovered, and the Quantikine Human Hepatocyte Growth Factor (HGF) ELISA kit (Funakoshi The amount of HGF in the medium was measured using the product manufactured by Shah, Code.RS-0641-00). After the medium was recovered, the cells were washed with PBS, and then 500 µl of cytolysis buffer (50 mM HEPES pH 7.4, 10 mM EDPA, 0.1% Triton x 100, 1 mM PMSF, 1 µg / ml pepstatin A, 1 µg / Ml leupetin). After sonication for more complete dissolution, the supernatant (cell extract) was prepared by centrifugation, and the amount of HGF in the cells was measured to be equal to the concentration of HGF in the medium. Kagome kelp-derived fucoidan described in Reference Example 1- (1) was added so as to have a final concentration of 10 µg / ml. Equivalent amount of distilled water was added as a negative control. The HGF concentration in the medium of the fucoidan addition group increased significantly more time-dependently than the negative control of distilled water addition. On the other hand, the amount of HGF in the cells of the fucoidan-added group decreased up to 4 hours after the addition, but after that, it became a constant low value. There was no such change in the negative control of distilled water addition, and it was always increasing. As a result, the fucoidan had an effect of releasing HGF from cells and promoting HGF production, and the production of HGF was found to increase with time. These results are shown in Tables 43-45.
[370] Changes in HGF Volume in Media Over Time00.5One24812244872 Production of HGF (ng / well) Control00.110.190.291.071.562.343.467.4510.5 Fucoidan addition0.510.461.232.864.006.709.1512.422.829.3
[371] Change of HGF amount in cells over timeIncubation time (hours) 00.5One24812244872 Production of HGF (ng / well) Control0.510.520.660.560.710.630.860.731.021.20 Fucoidan addition0.880.660.450.460.250.290.200.200.180.23
[372] Changes in Total HGF Content in Media and Cells Over TimeIncubation time (hours) 00.5One24812244872 Production of HGF (ng / well) Control0.510.630.850.841.782.193.194.198.4711.6 Fucoidan addition1.391.121.683.314.257.009.3512.622.929.5
[373] (3) 500 µl of MRC-5 cells (CCL 171: Dainippon Seiyaku, code. 02-021) suspended in DME medium containing 10% fetal bovine serum to 1 x 10 ⁵ cells / ml was prepared in 48 wells. Cell culture plates were placed and exchanged with DME medium containing 1% fetal bovine serum after 24 hours of incubation at 37 ° C. in the presence of 5% CO ₂ . Thereafter, the sample was added and cultured again for 0, 0.5, 1, 2, 4, 8, 12, 24, 48, 72 hours, and then the medium was recovered, and the Quantikine Human Hepatocyte Growth Factor (HGF) ELISA kit (Funakoshi The amount of HGF in the medium was measured using the product manufactured by Shah, Code.RS-0641-00). After the medium was recovered, the cells were washed with PBS, and 500 µl of cell lysis buffer (50 mM HEPES pH 7.4, 10 mM EDPA, 0.1% Triton × 100, 1 mM PMSF, 1 µg / ml pepstatin A, 1 µg / Ml leupetin). After sonication for more complete lysis, the supernatant (cell extract) was prepared by centrifugation, and the amount of HGF in the cells was measured to be equal to the concentration of HGF in the medium. 7-12SFd-F was added to a final concentration of 10 μg / ml. Equivalent amount of distilled water was added as a negative control. The HGF concentration in the medium of the 7-12SFd-F addition group increased significantly more time-dependently than the negative control of the distilled water addition. On the other hand, the amount of HGF in the cells of the 7-12SFd-F addition group decreased for a while after the addition, but changed to increase thereafter. There was no such change in the negative control of distilled water addition and was always constant. As a result, 7-12SFd-F has an effect of releasing HGF from cells and an activity of promoting high HGF production, and the production of HGF was shown to increase with time. After that, it became a constant low value. These results are shown in Tables 46-48.
[374] Changes in HGF Volume in Media Over TimeIncubation time (hours) 00.5One24812244872 Production of HGF (ng / well) Control0.140.330.360.631.291.602.543.897.9913.3 Add 7-12SFd-f0.481.822.162.533.124.015.849.8217.023.5
[375] Change of HGF amount in cells over timeIncubation time (hours) 00.5One24812244872 Production of HGF (ng / well) Control2.653.112.732.772.312.822.804.615.366.74 Add 7-12SFd-f2.791.781.651.311.061.311.071.562.663.09
[376] Changes in Total HGF Content in Media and Cells Over TimeIncubation time (hours) 00.5One24812244872 Production of HGF (ng / well) Control2.793.433.093.393.604.415.348.5013.320.2 Add 7-12SFd-f3.263.603.813.844.185.316.9111.419.726.6
[377] (4) 500 μl of MRC-5 cells (CCL 171: Dainippon Seiyaku, code. 02-021) suspended in DME medium containing 10% fetal bovine serum to 1 × 10 ⁵ cells / ml was prepared in 48 wells. Cell culture plates were placed and exchanged with DME medium containing 1% fetal bovine serum after 24 hours of incubation at 37 ° C. in the presence of 5% CO ₂ . Thereafter, the sample was added and incubated again for 24 hours. This medium was recovered and the amount of HGF in the medium was measured using a Quantikine Human Hepatocyte Growth Factor (HGF) ELISA kit (Funakoshisha, Code. RS-0641-00). In addition, after washing the cells with PBS, 500 μl of cytolysis buffer (50 mM HEPES pH 7.4, 10 mM EDPA, 0.1% Triton × 100, 1 mM PMSF, 1 μg / ml pepstatin A, 1 μg / ml leupetin) Dissolved in. After sonication for more complete lysis, the supernatant (cell extract) was prepared by centrifugation, and the amount of HGF in the cells was measured to be equal to the concentration of HGF in the medium. 7-12SFd-F was added at a final concentration of 1, 10, 100 μg / ml. Equivalent amount of distilled water was added as a negative control. All experiments were performed three times in succession, and the average value was adopted. As a result, as shown in Table 49, HGF in the medium of the 7-12SFd-F addition group significantly increased the HGF production in a concentration-dependent manner of 7-12SFd-F than the negative control of the distilled water addition. On the other hand, the amount of HGF in the cells decreased in a concentration-dependent manner of 7-12SFd-F. In addition, the amount of total HGF in intracellular and externally increased. As a result, it was shown that 7-12SFd-F had an effect of releasing HGF from cells and promoting HGF production. The results are shown in Table 49.
[378] HGF Production with 7-12SFd-F7-12SFd-F addition amount (final concentration µg / ml) 0One10100 Production of HGF (ng / well) Badge5.668.4617.122.0 In the cell6.286.044.151.59 Whole quantity11.914.521.323.6
[379] Example 6
[380] (1) 500 μl of MRC-5 cell solution suspended in DME medium containing 10% fetal bovine serum to 1 × 10 ⁵ cells / ml is placed in a 48 well cell culture plate, and is placed at 37 ° C. in the presence of 5% CO _2. After time incubation it was exchanged with DME medium containing 1% fetal bovine serum. Thereafter, cycloheximide (protein synthesis inhibitor: manufactured by Nakarai Tessha) was added so as to have a final concentration of 0, 1, 10 µg / ml, and further cultured for 24 hours after the addition of the test sample. This medium was recovered and the amount of HGF in the medium was measured using an HGF ELISA kit. Kagome kelp-derived fucoidan described in Reference Example 1- (1) was added so as to have a final concentration of 1, 10, 100 µg / ml. As a control, the same amount of distilled water as the sample was added. All experiments were carried out two times in succession and the average value was adopted. The results are shown in Table 50. In Table 50, the production of HGF of the control group was expressed as 100%.
[381] As shown in Table 50, by adding cycloheximide, the HGF concentration in the medium of the said fucoidan addition group decreases depending on cycloheximide concentration, and the inhibition rate is the inhibition by cycloheximide of the fucoidan no addition control group. It was inhibited depending on the concentration of cycloheximide to the same extent. From these, it became clear that protein synthesis is involved in inducing production of HGF by fucoidan.
[382] Kagome kelp-derived fucoidan (µg / ml)Cycloheximide addition amount (µg / mL) 0One10 Production to HGF ratio (%) Control1003527 One22611072 10317130110 100456206127
[383] (HGF production of the control was 7.3 ng / ml)
[384] (2) 500 μl of MRC-5 cells (CCL 171: Dainippon Seiyakusha, code. 02-021) suspended in DME medium containing 10% fetal bovine serum to 1 × 10 ⁵ cells / mL was used for 48 wells. Into a cell culture plate and replaced with DME medium containing 1% fetal bovine serum after incubation for 24 hours in the presence of 37 ° C., 5% CO ₂ . Thereafter, cycloheximide (protein synthesis inhibitor: manufactured by Nakarai Tessha) was added so as to have a final concentration of 0, 1, 10 μg / ml, and the sample was further added, followed by incubation for 24 hours. This medium was recovered and the amount of HGF in the medium was measured using a Quantikine Human Hepatocyte Growth Factor (HGF) ELISA Kit (Code. RS-0641-00, manufactured by Funakoshisha, Co., Ltd.). Furthermore, after washing the cells with PBS, 500 μl of cell lysis buffer (50 mM HEPES pH7.4, 10 mM EDTA, 0.1% Triton × 100, 1 mM PMSF, 1 μg / ml pepstatin A, 1 μg / ml leupetin )). To dissolve more completely, after sonication, supernatant (cell extract) was prepared by centrifugation, and the amount of HGF in the cells was measured to be equal to the concentration of HGF in the medium. The production of HGF was expressed as 100% negative control. The inhibition rate was based on the HGF production amount when only 7-12SFd-F of each concentration was added, and the inhibition rate (%) of the cycloheximide addition fraction was computed. 7-12SFd-F was added so that the final concentration might be 1, 10, 100 µg / ml. As a negative control, the same amount of distilled water as the sample was added. All experiments were performed three times in succession, and the average value was employ | adopted. As a result, as shown in Table 51 and Table 52, by adding cycloheximide, the HGF amount of the sum total in the cell and the medium in the medium of the 7-12SFd-F addition group was dependent on the cycloheximide concentration in both cases. It was evident that the induction of HGF production by 7-12SFd-F from these involved protein synthesis rather than the release of HGF from simple cells.
[385] Inhibition of HGF Production Induction (during medium) 7-12SFd-F addition amount (㎍ / mL)Cycloheximide addition amount (µg / mL) 0One10 Production rate of HGF:% / Inhibition rate when no cycloheximide is added to 100%:% 0100/040/6040/60 One124/061/5139/69 10216/088/5953/75 100265/0117/5674/72
[386] Inhibition of HGF production induction (sum in cells and medium) 7-12SFd-F addition amount (㎍ / mL)Cycloheximide addition amount (µg / mL) 0One10 Production rate of HGF:% / Inhibition rate when no cycloheximide is added to 100%:% 0100/031/6927/73 One104/031/7025/76 10128/038/7028/78 100137/047/6637/73
[387] (3) 500 μl of MRC-5 cells (CCL 171: Dainippon Seiyakusha, code. 02-021) suspended in DME medium containing 10% fetal bovine serum to 1 × 10 ⁵ cells / ml was used in 48 wells. Into a cell culture plate and replaced with DME medium containing 1% fetal bovine serum after incubation for 24 hours in the presence of 37 ° C., 5% CO ₂ . Thereafter, ectinomycin D (RNA synthesis inhibitor: manufactured by Sigma Co., Ltd.) was added so as to have a final concentration of 0, 0.1, and 1 µg / ml, and the sample was further added, followed by incubation for 24 hours. This medium was recovered and the amount of HGF in the medium was measured using a Quantikine Human Hepatocyte Growth Factor (HGF) ELISA Kit (Code. RS-0641-00, manufactured by Funakoshisha, Co., Ltd.). The production amount of HGF was expressed as 100% of the negative control. Inhibition rate calculated the inhibition rate (%) of the actinomycin D addition fraction on the basis of the production amount of HGF when only fucoidan of each concentration was added. The fumeidan derived from Kagome kelp as described in Reference Example 1- (1) was added so as to have a final concentration of 1, 10, 100 µg / ml. As a negative control, the same amount of distilled water as the sample was added. All the experiments were performed two times continuously and the average value was employ | adopted. As a result, as shown in Table 53, by adding the actinomycin D, the HGF concentration in the medium of the fucoidan-added group was inhibited depending on the actinomycin D concentration. From these, it was suggested that RNA synthesis may be involved in inducing production of HGF by fucoidan, and it was clear that HGF was not free from simple cells.
[388] Kagome kelp-derived fucoidan (µg / ml)Actinomycin D added amount (µg / ml) 00.1One Production of HGF:% / Inhibition rate when 100% of no tinomycin D was added:% 0100/079/2183/17 One244/0138/43119/51 10343/0224/35239/30 100492/0341/31285/42
[389] (4) 500 μl of MRC-5 cells (CCL 171: Dainippon Seiyakusha, code. 02-021) suspended in DME medium containing 10% fetal bovine serum to 1 × 10 ⁵ cells / mL was used for 48 wells. Into a cell culture plate and replaced with DME medium containing 1% fetal bovine serum after incubation for 24 hours in the presence of 37 ° C., 5% CO ₂ . Thereafter, ectinomycin D (RNA synthesis inhibitor: manufactured by Sigma Co., Ltd.) was added so as to have a final concentration of 0, 0.1, and 1 µg / ml, and the sample was further added, followed by incubation for 24 hours. This medium was recovered and the amount of HGF in the medium was measured using a Quantikine Human Hepatocyte Growth Factor (HGF) ELISA Kit (Code. RS-0641-00, manufactured by Funakoshisha, Co., Ltd.). In addition, the cells were washed with PBS and then in 500 µl of cell lysis buffer (50 mM HEPES pH7.4, 10 mM EDTA, 0.1% TritonX100, 1 mM PMSF, 1 µg / ml pepstatin A, 1 µg / ml leupetin). Dissolved. To dissolve more completely, after sonication, supernatant (cell extract) was prepared by centrifugation, and the amount of HGF in the cells was measured to be equal to the concentration of HGF in the medium. The production of HGF was expressed as 100% negative control. The inhibition rate was based on the HGF production amount when only 7-12SFd-F of each concentration was added, and the inhibition rate (%) of the actinomycin D addition fraction was computed. 7-12SFd-F was added so that the final concentration might be 1, 10, 100 µg / ml. As a negative control, the same amount of distilled water as the sample was added. All experiments were performed three times in succession, and the average value was employ | adopted. As a result, as shown in Table 54 and Table 55, by adding the actinomycin D, the total amount of HGF in the cells and the medium in the medium of the 7-12SFd-F addition group was dependent on the concentration of the actinomycin D. Was inhibited. From these, it was suggested that RNA synthesis may be involved in inducing production of HGF by 7-12SFd-F, and it was clear that HGF was not free from simple cells.
[390] Inhibition of HGF Production Induction (during medium) 7-12SFd-F addition amount (㎍ / mL)Actinomycin D added amount (µg / ml) 00.1One Production of HGF:% / Inhibition rate when 100% of no tinomycin D was added:% 0100/076/2480/20 One130/095/2796/26 10225/0182/19152/32 100295/0212/28187/48
[391] Inhibition of HGF production induction (sum in cells and medium) 7-12SFd-F addition amount (㎍ / mL)Actinomycin D added amount (µg / ml) 00.1One Production of HGF:% / Inhibition rate when 100% of no tinomycin D was added:% 0100/061/3959/41 One98/064/3561/38 10113/083/2771/37 100126/091/2881/36
[392] Example 7
[393] (1) Partial liver resection was performed by surgical treatment using 7-week-old male Wistar rats as follows. That is, rats were opened under ether anesthesia, and about 30% of the liver was ligated with root sutures with a surgical suture and then excised. The open abdomen was closed with a suture needle.
[394] Kagome kelp-derived fucoidan described in Reference Example 1- (1) was administered intraperitoneally at 12-hour intervals immediately after resection. In the control group, saline was administered intraperitoneally.
[395] At 24 hours or 72 hours after liver resection, rats were bled from the abdominal aorta under anesthesia and the plasma with 0.1% ethylenediamine tetraacetate diacetate was centrifuged. The amount of HGF in plasma was measured using an HGF ELISA kit (manufactured by Tokushu Meneki Genkyusho Co., Ltd.).
[396] The results are shown in Table 56. The numbers in the table indicate the mean ± standard error, and () represents the number of rats per group. In addition, * in the table means having a significant difference with a risk of 5% or less compared to the control.
[397] Dosing sampleDose (mg / kg)HGF concentration in plasma (ng / ml) 24 hours72 hours Physiological Saline (Control) 0.28 ± 0.04 (4)0.30 ± 0.02 (3) Fucoidan from Kagome Kelp0.110.40 ± 0.12 (4) 0.65 ± 0.18 (4)0.52 ± 0.06 (5) * 0.64 ± 0.09 (2) *
[398] Compared with the control group, the fucoidan-administered group was found to have an increase in the amount of HGF in plasma 24 hours after liver resection, and a significant increase was found after 72 hours.
[399] As described above, fucoidan promotes rapid regeneration after surgery in liver disease requiring surgical operation and is useful for restoring liver function by inducing HGF production.
[400] (2) Partial liver resection was performed by surgical treatment using 7-week-old male Wistar rats. The abdomen was opened under ether anesthesia, and about 30% of the liver was excised after ligation of the root vessel with a surgical suture. The open abdomen was closed with a suture needle. Enzyme-treated F-fucoidan prepared in Reference Example 2- (4) was taken immediately after the first removal, and divided into two tides and orally administered. Physiological saline was administered to the control group. After 24 hours of liver resection, rats were bled from the aorta under anesthesia and the plasma with 0.1% ethylenediamine tetrasodium acetate was centrifuged. The amount of HGF in plasma was measured using an HGF ELISA kit (Tokushu Meneki Genkyusho).
[401] The results are shown in Table 57. The numbers in the table indicate the mean ± standard error, and () indicates the number of rats per group. In addition, * in the table means a group having a significant difference with a risk of less than 1% in fertilizer with the control.
[402] groupHGF concentration in plasma (ng / ml) 24 hours Saline solution0.184 ± 0.33 (6) Enzyme treatment F-fucoidan (1 g / kg / day)0.505 ± 0.97 * (5)
[403] The enzyme-treated F-fucoidan-administered group found a significant elevation after 24 hours of liver resection compared to the control.
[404] As described above, Fucoidan and 7-12SFd-F-rich F-fucoidan induce HGF production, which promotes rapid regeneration after surgery in liver disease requiring surgical surgery, and is useful for restoring liver function.
[405] Example 8
[406] DMEM medium containing Hs68 cells (ATCC CRL-1635), a human neonatal foreskin epithelial cell line expressing h-IGF-1, a type of proliferative factor such as insulin, containing 10% fetal bovine serum (FBS: manufactured by BioWhitaker) (Made by Gibco BRL Co., Ltd.), incubated at 37 ° C. in the presence of 5% CO ₂ until the cells are saturated in the incubator, and 3 × 10 ³ cells / well with trypsin-EDTA solution (manufactured by Bio Whitaker). Suspended in the medium so as to separate, and 200 µl of each well of a 96 well microtiter plate was injected. After 5 to 7 days of culture, the medium was discarded almost at the time when the cells became saturated in the incubator, and the I fraction described in Reference Example 1- (2) of 0, 12.3, 37, 111, 333, 1000, or 3000 µg / ml. 200 μl / well of the medium containing, II fraction, III fraction, or Kagome Kelp-derived fucoidan described in Reference Example 1- (1) was added. The culture supernatant was recovered over time at 1, 4, 12, and 24 hours by taking a 24-hour time course, and h-IGF-1 ELISA kit (Diagnostics, Inc. Production). The results are shown in Tables 58 to 61. In addition, the control is sample free.
[407] Kagome kelp-derived fucoidan (µg / ml)H-IGF-1 concentration in medium (ng / ml) 1 hours4 hours12 hours24 hours contrast4.32.94.14.5 12.322.914.710.511.8 3717.513.910.811.4 11114.613.710.37.8 33313.817.69.47.7 100013.914.714.67.5 300015.914.014.07.2
[408] I fraction (μg / ml)H-IGF-1 concentration in medium (ng / ml) 1 hours4 hours12 hours24 hours contrast6.85.45.05.0 12.313.910.57.67.3 3719.011.78.310.1 11120.411.09.59.9 33318.712.210.910.3 100017.713.212.711.3 300019.012.113.111.7
[409] Ⅱ fraction (µg / ml)H-IGF-1 concentration in medium (ng / ml) 1 hours4 hours12 hours24 hours contrast4.32.94.14.5 12.318.510.26.97.0 3720.19.99.29.2 11120.011.19.88.9 33316.312.79.79.2 100017.012.210.08.8 300017.911.310.07.6
[410] Ⅲ fraction (µg / ml)H-IGF-1 concentration in medium (ng / ml) 1 hours4 hours12 hours24 hours contrast4.32.94.14.5 12.316.510.98.58.6 3716.710.211.18.4 11111.811.58.66.9 3339.611.28.05.9 10007.910.410.66.6 30007.19.49.76.2
[411] As shown in Tables 58 to 61, Kagome Kelp-derived fucoidan, I fraction, II fraction and III fraction showed h-IGF-1 production inducing activity. h-IGF-1 production inducing activity peaked at 1 hour by the addition of 12 to 100 µg / ml of the sample. In addition, toxicity and proliferation inhibitory activity against Hs68 cells in each sample were not recognized. Other acid polysaccharides, their degradation products, acid oligosaccharides, acidic monosaccharides and salts thereof described in Reference Examples were similarly recognized for h-IGF-1 production inducing activity.
[412] Example 9
[413] Rat fibroblast LM cells (ATCC CCL-1.2) were suspended in 1.5 × 10 ⁵ cells / ml in M119 medium (manufactured by ICN) containing 0.5% of bactopeptone (manufactured by Difco), and then 0.1 ml each in a 96 well plate. Sprinkle and incubate aseptically.
[414] After incubation for 3 days, the medium was removed and converted to M199 medium containing 0.5% fetal bovine albumin (manufactured by Sigma). Here, the example of Kagome kelp-derived fucoidan described in Reference Example 1- (1) was added so as to have a final concentration of 0, 62.5, 250, and 1000 µg / ml, and cultured for 24 hours. Distilled water was added as a control. After the completion of the culture, the concentration of NGF in the culture solution was measured by an Enzyme immunoassay method (NGF Emax Immuno Assay System: manufactured by Promega). The production amount of NGF was expressed as 100% of NGF production of the control group. All experiments were carried out two times in succession and the average value was adopted. As a result, it is shown in Table 62. As shown in Table 62, Kagome kelp-derived fucoidan promoted nerve growth factor production of L-M cells in a concentration-dependent manner. The fractions also showed the same activity. In addition, other acidic polysaccharides, their degradation products, acidic oligosaccharides, acidic monosaccharides and salts thereof described in Reference Examples also exhibited the same NGF production inducing action.
[415] sampleConcentration (µg / ml)% Increase in NGF production Kagome kelp-derived fucoidan62.5117 250166 1000179
[416] (NGF production of control was 155 pg / ml)
[417] Similarly, the promoting activity of nerve growth factor production was measured about the I fraction, II fraction, and III fraction of Reference Example 1- (2), and activity was recognized for each fraction. The results are shown in Table 63. In addition, other acidic polysaccharides, their degradation products, acidic oligosaccharides, acidic monosaccharides and salts thereof described in Reference Examples also exhibited NGF production inducing action.
[418] sampleConcentration (µg / ml)% Increase in NGF production I fraction250505.6 500619.6 1000806.5 Ⅱ fraction250664.5 500864.5 10001137.4 Ⅲ fraction2501021.1 5001187.0 10001265.0
[419] (NGF production of control was 50.03 pg / ml)
[420] Example 10
[421] (1) Male C / 3H / He mice were purchased from Nihon SLC and used for experiments from 5 weeks of age after preliminary breeding. Kagome kelp-derived fucoidan prepared in Reference Example 1- (1) was suspended and dissolved in ethanol at a concentration of 3%, and 200 µl per animal was applied to the back of the mouse. Ethanol was applied equally to the control group. Administration was carried out once a day, and eight days a day. On the 9th day of the start of administration, the skin was peeled off, and HGF activity in the skin was measured by an ELISA kit (Tokushu Meneki Genkyu Sho).
[422] The results are shown in Table 64. The numbers in the table indicate the mean ± standard error of five examples.
[423] HGF activity extracted from the skin was significantly higher than that of the control group in the fucoidan application group, and the action of inducing HGF production by fucoidan application was recognized.
[424] HGF activity in skin (ng / g tissue) Control group (N = 5)16.31 ± 2.86 Fucoidan-coated group (N = 5)104.46 ± 4.05
[425] Standard value ± Mean error
[426] (2) The lotion of the present invention described in Example 26- (1) described below was compared with a control lotion containing no fucoidan, and 25 sensory adult women aged 20 to 35 years were subjected to a sensory test blindly. As a result, the arguments determined to be more effective are shown in Table 65.
[427] Skin moistureThe softness of the skinSkin elasticity Lotion of the present invention211916 Lotion of contrast569
[428] As a result, it was shown that the skin lotion of the present invention of the fucoidan blend has excellent skin moisturity, softness and elasticity due to HGF production inducing action of fucoidan.
[429] Example 11
[430] (1) 98 mg of F-fucoidan prepared by the method described in Reference Example 1- (2) was dissolved in 5 ml of DMSO, and 980 mg of piperidine sulfate was added at room temperature, followed by stirring at 80 ° C for 2 hours. The reaction solution was cooled and then dialyzed with a dialysis membrane having a molecular weight of 1000 cuts for 2 days. The obtained dialysis internal liquid was supplied to a cation exchange column [Amberlite IRA-120 (Na ⁺ )], and 98 mg of high-sulfur oxides of F-fucoidan were prepared by drying under reduced pressure.
[431] (2) 34 mg of 7-12SFd-F prepared by the method of Reference Example 2 was dissolved in 4 ml of DMSO, and then the high sulfur oxide of 7-12SFd-F 98 was obtained in the same manner as in Example 11- (1). Mg was prepared.
[432] (3) High-sulfur oxides of F-fucoidan prepared in Example 11- (1) (sample ①) and 7-12SFd-F-high sulfur oxides prepared in Example 11- (2) in the same manner as in Example 1. HGF production-inducing activity of (sample ②), F-fucoidan (sample ③) prepared in Reference Examples 1- (2), and 7-12SFd-F (sample 4) prepared in Reference Example 2 was examined. Each sample was added to a final concentration of 1, 10, 100 μg / ml. As a control, the same amount of distilled water as the sample was added.
[433] The results are shown in Table 66. In Table 66, the HGF production of the control group was expressed as 100%. All experiments were carried out two times in succession and the average value was adopted. As shown in Table 66, samples ① to ④ induced the production of HGF. Also, in the high sulfur oxides, its HGF production inducing activity was higher than that of the unhigh sulfur oxidation treatment. From this, it was evident that the natural sulfated sugars already present were also subjected to sulfate treatment to enhance the HGF production inducing activity.
[434] In addition, the amount of sulfuric acid content was determined by heating a 0.2 ml (1 to 10 mg / ml) solution of 1 N HCl of each sample at 105 ° C. for 4 hours, in which 1.9 ml of 0.1 N HCl solution and 1% barium chloride were added to 0.1 ml. 0.25 ml of -0.5% gelatin solution was added and left to stand for 20 minutes, followed by measurement of absorbance at 500 nm. Calibration curve was determined 0, 1, 3, 5, 7, 10, 15, 20 mM sodium sulfate, 1 N HCl solution of the standard sample to create and sulfate content of the respective samples (in terms of SO ₃₎ from the calibration curve as. This calibration curve is shown in Fig. 2 and the sulfuric acid content of each sample is shown in Table 67.
[435] Concentration (µg / ml)Production of HGF (%) Sample ①Sample ②Sample ③Sample ④ One346192312192 10715214493229 100794499598355
[436] (HGF production of control was 8.15 ng / ml)
[437] sampleSulfuric acid content (SO ₃ equivalent,%) F-Pucoidan40 High Sulfur Oxides of F-fucoidan47 7-12SFd-F40 High Sulfur Oxide of 7-12SFd-F48
[438] Example 12
[439] NHDF cells (human normal skin fibroblasts, manufactured by Bio Wittaker) cultured in DMEM medium containing 10% fetal bovine serum were suspended in DMEM medium containing 10% fetal bovine serum to 1 x 10 ⁵ cells / ml. , 500 μl each was put in a 48 well cell culture plate and incubated for 24 hours. Thereafter, the mixture was replaced with DMEM medium containing 1% fetal bovine serum, and 10 nM tetradecanoyl phorbol 13-acetate (TPA: manufactured by Gibco BRL) and a sample were added. After incubation for 20 hours after the addition, the medium was recovered, and the amount of HGF in the medium was measured using a Quantikine Human Hepatocyte Growth Factor (HGF) ELISA Kit (Code. RS-0641-00, manufactured by Funakoshisha, Co., Ltd.). The production amount of HGF was expressed with the negative control as 100%. The samples were added so that their final concentrations were 1, 10 and 100 µg / ml of fucoidan described in Reference Example 1- (1). Heparin was added to 1, 10 µg / ml. As a negative control, the same amount of distilled water as the sample was added. Cultivation of the induction experiment was carried out by adding 10 nM TPA simultaneously with addition of the sample. All the experiments were performed three times consecutively, and the average value was employ | adopted. The results are shown in Table 68. All of the cell groups added with fucoidan significantly increased HGF production than the negative control of distilled water addition. In addition, the production of HGF was significantly higher than that of heparin. Accordingly, it was shown that the fucoidan described in Reference Example 1- (1) has an activity of promoting the production of HGF higher than heparin, which has been confirmed to induce HGF.
[440] Concentration (µg / ml)HeparinFucoidan 0100100 One9501140 1021702470 100-2770
[441] (However, HGF production of the control group was 0.20 ng / ㎖)
[442] Example 13
[443] DMEM containing 10% fetal bovine serum so that Hs68 cells (human neonatal foreskin cells (ATCC CRL-1635, manufactured by Dainippon Seiyakusha)) grown in DMEM medium containing 10% fetal bovine serum to 1 x 10 ⁵ cells / ml Suspended in the medium, 500 μl each put into a 48 well cell culture plate and incubated for 24 hours. Thereafter, it was exchanged with DMEM medium containing 1% fetal bovine serum, and 10 nM TPA and a sample were added. In addition, the division which adds only a sample without adding TPA was performed similarly. The medium was recovered and the amount of HGF in the medium was measured using a Quantikine Human Hepatocyte Growth Factor (HGF) ELISA Kit. Furthermore, after washing the cells with PBS, 500 μl of cell lysis buffer (50 mM HEPES pH7.4, 10 mM EDTA, 0.1% Triton × 100, 1 mM PMSF, 1 μg / ml pepstatin A, 1 μg / ml leupetin )). To dissolve more completely, after sonication, supernatant (cell extract) was prepared by centrifugation, and the amount of HGF in the cells was measured to be equal to the concentration of HGF in the medium. 7-12SFd-F prepared in Reference Example 2- (3) was added so as to have a final concentration of 0.1, 1, 10, 100 µg / ml. As a negative control, the same amount of distilled water as the sample was added. All experiments were performed three times in succession, and the average value was employ | adopted. The results are shown in Tables 69 to 71. Significant production of HGF with no TPA was not recognized. However, when TPA was added, the amount of HGF in the cells decreased in a concentration-dependent manner of 7-12SFd-F, the amount of HGF and total HGF in the medium increased in a concentration-dependent manner of 7-12SFd-F, and also total HGF. The amount was significantly increased than the control group without addition. In addition, the increase in the amount of HGF when the mRNA was increased was very remarkable compared to the case where the amount of mRNA was small in the TPA treatment. From this, it became clear that 7-12SFd-F promoted mRNA transcription and remarkably promoted the release and production of HGF when a large amount of HGF was required.
[444] HGF amount in Hs68 / 7-12SFd-F medium (pg / Well) 7-12SFd-F (μg / ml)No TPA10 nMTPA 0N.D.65.54 0.1N.D.71.04 OneN.D.99.25 10N.D.226.14 100N.D.260.49
[445] N.D. Indicates below the detection limit.
[446] HGF content in Hs68 / 7-12SFd-F cells (pg / Well) 7-12SFd-F (μg / ml)No TPA10 nMTPA 098.62155.65 0.187.60151.99 One62.47142.17 10N.D.120.70 100N.D.95.67
[447] N.D. Indicates below the detection limit.
[448] Hs68 / 7-12SFd-F Total HGF Volume (pg / Well) 7-12SFd-F (μg / ml)No TPA10 nMTPA 0 221.19 0.1 223.02 One 241.42 10 346.84 100 356.05
[449] Example 14
[450] MRC-5 cells (CCL 171: Dainippon Seiyakusha Code. 02-021) incubated in DMEM medium containing 10% fetal bovine serum contained 10% fetal bovine serum to 2.5 x 10 ⁵ cells / ml. It was suspended in DMEM medium, put into a 6 well cell culture plate, and cultured for 24 hours at 37 degreeC in presence of 5% carbon dioxide gas. Thereafter, the cells were replaced with DMEM medium containing 1% fetal bovine serum, and cultured again for 22 hours. Then, a final concentration of 100 ㎍ / ㎖ is such that in Reference Example 2- (3) was prepared so that the LM heparin 7-12SFd-F, 1 ㎍ / ㎖ in (Celsus laboratories Sha, Ltd.), so that the dimethyl 1 μ M Each culture solution was added with prostaglandin E ₁ (PGE ₁ ) (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in sulfoxide (DMSO). As a control, a medium to which DMSO was added was used. In addition, the solvent of each said additive was added so that it might become 1%. Incubation was carried out again, and all RNAs were extracted at 0, 2, 4, 6, 8, 10, 12 and 24 hours. RNeasy Mini Kit (made by QIAGEN) was used for extraction of all RNA. RT-PCR is RNA PCR Kit ver. 2.1 (Takara Shujo Co., Ltd., R019A) was used. The reverse transcription reaction was subjected to heat denaturation treatment on all RNAs, followed by 10 minutes at 30 ° C, 30 minutes at 42 ° C, and 5 minutes at 99 ° C using a random primer (N6) (manufactured by Takara Shuzo, 3801). In order to detect mRNA of HGF, the primer of SEQ ID NO: 1 of the sequence table was used as a sense primer, and the primer of SEQ ID NO: 2 of the sequence table was used as an antisense primer. The product amplified by this primer is 415 bp. In addition, in order to perform a semi-quantitative experiment, the housekeeping gene β-actin was also detected. The primer described in SEQ ID NO: 3 of the sequence table was used as the sense primer, and the primer described in SEQ ID NO: 4 of the sequence table was used as the antisense primer. The product amplified by this primer is 275 bp. PCR was performed by PJ9600 (Perkin Elmer). The PCR cycle was subjected to thermal denaturation at 94 ° C for 2 minutes, followed by 24 cycles of thermal denaturation at 94 ° C for 30 seconds, annealing at 59 ° C for 30 seconds, and extension reaction at 72 ° C for 60 seconds. After the reaction, 2% agarose gel electrophoresis and ethidium bromide staining were performed, and the gel was observed under UV irradiation. HGF mRNA was detected in all samples. Induction of mRNA by PGE ₁ was recognized as compared to the control, but induction of mRNA by 7-12SFd-F and LM heparin was not recognized. From this, it is evident that in the state of always transcribing the mRNA of HGF, the promotion of the transcription of HGF mRNA by addition of 7-12SFd-F and LM heparin does not occur, and the excess of 7-12SFd-F and LM heparin is excessive. It has been clarified that no production of HGF occurs.
[451] Example 15
[452] Induction of HGF production by 7-12SFd-F in NHDF cells in the same manner as in Example 13 except that NHDF cells (human normal skin fibroblasts: manufactured by Bio Whittaker) were used instead of the HS68 cells used in Example 13. The activity was investigated. The results are shown in Tables 72 to 74.
[453] In the absence of TPA, the amount of HGF in the cells decreased in a concentration-dependent manner of 7-12SFd-F, the amount of HGF and total HGF in the medium increased in a concentration-dependent manner of 7-12SFd-F, and the amount of total HGF was 7-12SFd-. Significantly increased than the control group without F. From this, it became clear that even when there was little mRNA of HGF such as TPA untreated, the release of HGF on the cell surface and the synthesis of HGF were promoted. In addition, when TPA was added, the HGF content in the cells decreased in a concentration-dependent manner of 7-12SFd-F, and the HGF amount and total HGF content in the medium increased in a concentration-dependent manner of 7-12SFd-F. The amount of HGF was significantly increased than the control group without 7-12SFd-F. In addition, the increase in the amount of HGF when the mRNA was increased in this manner was very remarkable as compared with the case where the amount of mRNA was small in the absence of TPA. From this, 7-12SFd-F promotes the release and production of HGF to a lesser extent when the amount of mRNA is small, and also promotes the release and production of HGF when a large amount of HGF is required. Significant facilitating became clear.
[454] HGF amount in NHDF / 7-12SFd-F medium (ng / Well) 7-12SFd-F (μg / ml)No TPA10 nMTPA 0N.D.0.39 One0.050.68 100.181.22 1000.3851.625
[455] N.D. Indicates below the detection limit.
[456] HGF content in NHDF / 7-12SFd-F cells (ng / Well) 7-12SFd-F (μg / ml)No TPA10 nMTPA 00.1850.24 One0.1450.19 100.0750.12 1000.0250.055
[457] NHDF / 7-12SFd-F Total HGF Volume (ng / Well) 7-12SFd-F (μg / ml)No TPA10 nMTPA 0 0.63 One0.1950.87 100.2551.335 1000.411.68
[458] Example 16
[459] NHDF cells (human normal dermal fibroblasts) incubated in DMEM medium containing 10% fetal bovine serum were suspended in DMEM medium containing 10% fetal bovine serum to 2.5 x 10 ⁵ cells / ml. The wells were placed in a cell culture plate and incubated at 37 ° C. for 24 hours in the presence of 5% carbon dioxide gas. Thereafter, the mixture was replaced with DMEM medium containing 1% fetal bovine serum, and 7-12SFd prepared in 10 nM tetradecanoyl phorbol 13-acetate (TPA: manufactured by Gibco BRL) and Reference Example 2- (3). -F was added to a final concentration of 100 μg / ml. In addition, the division which adds only 7-12SFd-F without adding TPA was similarly performed. In addition, all RNAs were extracted at 4, 6, 8 and 10 hours. RNeasy Mini Kit (QIAGEN) was used for extraction of all RNA. RT-PCR was carried out similarly to Example 14 except having made 28 cycles of PCR. After the reaction, 2% agarose gel electrophoresis and ethidium bromide staining were performed, and the gel was observed under UV irradiation.
[460] As a result, in the case of no TPA addition, the amount of transcription of HGF mRNA was very small, but a slight increase in mRNA transcription amount was found 4 hours after the addition by addition of 7-12SFd-F. On the other hand, by the addition of TPA, the amount of HGF mRNA significantly increased at any time. When TPA and 7-12SFd-F were added, it was clear that the amount of HGF mRNA increased after 4 hours compared to the case where only TPA was added.However, at 6 hours, the difference due to the addition of 7-12SFd-F was increased. Was not. That is, it became clear that 7-12SFd-F markedly promoted transcription in the early stage when mRNA was started to be actively carried out by requiring HGF, but then the promoting effect was lost. From this, it became clear that 7-12SFd-F induces HGF production at the moment when HGF is needed, and thereafter, does not induce excess HGF production.
[461] Example 17
[462] HL60 cells (human progenitor leukemia cells) cultured in RPMI 1640 medium containing 10% fetal bovine serum were suspended in RPMI 1640 medium containing 1% fetal bovine serum to 5 x 10 ⁵ cells / ml, and 500 µl. Each was put into a 48 well cell culture plate. Thereafter, 10 nM TPA was added, and the sample was added again. Incubated for 24 hours after addition. In addition, the division which adds only a sample without adding TPA was performed similarly. This medium was recovered and the amount of HGF in culture was measured using a Quantikine Human Hepatocyte Growth Factor (HGF) ELISA Kit. Furthermore, after washing the cells with PBS, 500 μl of cell lysis buffer (50 mM HEPES pH7.4, 10 mM EDTA, 0.1% Triton × 100, 1 mM PMSF, 1 μg / ml pepstatin A, 1 μg / ml leupetin )). To dissolve more completely, after sonication, the cells were centrifuged to prepare a supernatant (cell extract), and the amount of HGF in the cells was measured in the same manner as the concentration of HGF in the medium. 7-12SFd-F prepared in Reference Example 2- (3) was added so as to have a final concentration of 1, 10, 100 µg / ml. As a negative control, the same amount of distilled water as the sample was added. All experiments were performed three times in succession, and the average value was employ | adopted. The results are shown in Tables 75 to 77.
[463] In the absence of TPA, the amount of HGF in the cells was not changed by the concentration of 7-12SFd-F. The amount of HGF in the medium was also increased to 100 µg / ml, but there was no significant change by the concentration of 7-12SFd-F. In addition, when TPA was added, the amount of HGF in the cells was not changed by the concentration of 7-12SFd-F, but was low overall. On the other hand, the amount of HGF and total HGF in the medium was significantly increased depending on the concentration of 7-12SFd-F, and the total HGF was significantly increased than the control group without the addition of 7-12SFd-F. In addition, the increase in the amount of HGF when such mRNA was increasing was very remarkable compared to the case where the amount of mRNA was small in the absence of TPA. From this, it became clear that 7-12SFd-F promoted mRNA transcription and remarkably promoted HGF release and production when a large amount of HGF was required.
[464] HGF amount in HL60 / 7-12SFd-F medium (pg / Well) 7-12SFd-F (μg / ml)No TPA10 nMTPA 076.74261.60 One67.10295.94 1078.54464.55 100162.85843.84
[465] HGF content in HL60 / 7-12SFd-F cells (pg / Well) 7-12SFd-F (μg / ml)No TPA10 nMTPA 0294.1283.37 One276.6789.39 10236.3067.09 100257.0485.77
[466] HL60 / 7-12SFd-F Total HGF Volume (pg / Well) 7-12SFd-F (μg / ml)No TPA10 nMTPA 0370.85344.97 One343.77385.32 10314.84531.64 100420.26929.60
[467] Example 18
[468] HL60 cells (human progenitor leukemia cells) cultured in RPMI 1640 medium containing 10% fetal bovine serum were suspended in RPMI 1640 medium containing 1% fetal bovine serum to 5 x 10 ⁵ cells / ml, and 2 ml Each well was put in a 6 well cell culture plate. Thereafter, 10 nM TPA and 7-12SFd-F prepared in Reference Example 2- (3) were added so as to have a final concentration of 100 µg / ml. In addition, the division which adds only 7-12SFd-F without adding TPA was similarly performed. In addition, all RNAs were extracted at 4, 6, 8 and 10 hours. RNeasy Mini Kit (made by QIAGEN) was used for extraction of all RNA. It carried out similarly to Example 14 except having made RT-PCR cycle into 32 cycles. After the reaction, 2% agarose gel electrophoresis and ethidium bromide staining were performed, and the gel was observed under UV irradiation.
[469] As a result, in the absence of TPA, the amount of transcription of HGF mRNA was very small, but a slight increase in the amount of HGF mRNA was found 4 hours after addition by addition of 7-12SFd-F. On the other hand, the addition of TPA increased the amount of HGF mRNA significantly at any time. When TPA and 7-12SFd-F were added, it became clear that the amount of HGF mRNA increased at 4 hours after addition compared to the case where only TPA was added, but at 6 hours by adding 7-12SFd-F. There was no difference. That is, it became clear that 7-12SFd-F required HGF and markedly promoted its transcription at an early stage when RNA transcription began to be actively performed, but then the promoting effect was lost. From this, it became clear that 7-12SFd-F induced HGF production at the moment when HGF was needed, and thereafter, did not induce excess HGF production.
[470] Example 19
[471] (1) Commercially available mugwort was lyophilized to obtain a mugwort lyophilized product. 10 g of wormwood powder which pulverized this mugwort freeze-dried product was suspended in 100 ml of chloroform, and the operation of filtering and recovering an insoluble fraction was repeated three times. Thereafter, the procedure of suspending and filtering in 100 ml of ethanol and recovering the insoluble fraction was repeated three times. Ethanol was completely removed from the insoluble fraction obtained in this operation, and suspended in 100 ml of distilled water. After keeping this suspension at 60 degreeC for 1 hour, it filtered. 2.5 times the amount of ethanol was added to the filtrate, cooled to -20 ° C, and then centrifuged at low temperature to obtain a precipitate. This precipitate was dissolved in distilled water, and freeze-dried to obtain a fraction of powdery and wormwood containing powdery sugar.
[472] (2) In the same manner as in Example 1- (1), the HGF production-inducing activity of the mugwort extract prepared in Example 19- (1) was examined. Samples were added to final concentrations of 1, 10 and 100 μg / ml. Equivalent amount of distilled water was added as a negative control. The production of HGF was expressed as 100% negative control. The results are shown in Table 78. All experiments were carried out two times in succession and the average value was adopted. As shown in Table 78, mugwort extract induced the production of HGF.
[473] Garland chrysanthemum extract (㎍ / mL)Production of HGF (%) One125 10148 100314
[474] (However, the HGF production of the control group was 8.21 ng / ㎖)
[475] Example 20
[476] In the same manner as in Example 1- (1), HGF production-inducing activity of the mugwort supernatant fraction prepared in Reference Example 13- (5) was measured. However, mugwort supernatant fraction was added with 1/1000 of the amount of medium. The results are shown in Table 79. As a result, it became clear that the mugwort extract has HGF production inducing activity even in fractions which do not precipitate in ethanol precipitation. From this, it was thought that there was activity even in the low molecular fraction which does not precipitate by ethanol precipitation.
[477] Mugwort supernatantProduction of HGF (%) 0100 1/1000 addition463
[478] (However, HGF production of the control group was 4.31 ng / ㎖)
[479] Example 21
[480] (1) 50 g of dried mugwort leaves (released by Sakamoto Kanboto) were placed in a homogenizer (manufactured by Nippon Seikisha), homogenized at 500 ml with acetone for 10 minutes, and filtered through a filter paper to obtain a residue. . The residue of 100 g of mugwort leaves obtained by performing the above operation twice was put into a homogenizer, added to 500 ml of acetone, homogenized at 8000 rpm for 10 minutes, and filtered with a filter paper to obtain a residue. This operation was repeated 4 times to obtain an acetone washing residue. The acetone washing residue was washed four times with 90% ethanol and four times with 80% ethanol to obtain the same as the acetone washing, and an ethanol washing residue was obtained. The above operation was performed once again from the beginning, and it combined and obtained 200 g of ethanol washing residue of the wormwood leaves.
[481] (2) To the ethanol washing residue, 30 mM phosphate buffer (pH 8.0) containing 10 liters of 100 mM sodium chloride and 10% ethanol was added, stirred at room temperature for 19 hours, filtered through a filter paper and the crude extract (filtrate) ) The crude extract thus obtained was concentrated to 2 liters with an ultrafiltration apparatus equipped with a holofiber having an exclusion molecular weight of 10,000, and then ultrafiltration while adding 100 mM sodium chloride containing 20 liters of 10% ethanol. Thereafter, the mixture was concentrated to 668 mL, 1 g of activated carbon was added thereto, stirred at room temperature for 30 minutes, and then centrifuged at 10000 rpm for 40 minutes to remove activated carbon. Trace amounts of activated carbon remaining in the supernatant were removed using a No. 5c filter. 66.8 mL was taken from the activated carbon treatment liquid, sufficiently dialyzed with distilled water, and lyophilized to obtain 670 mg of dried product. The dried product was named Mugwort Polymer Fraction (YPS). The remaining 601.2 ml of activated carbon treated solution was placed in an ultrafiltration apparatus and solvent-substituted with 10 mM imidazole hydrochloride buffer (pH7.0) containing 10% ethanol and 50 mM sodium chloride to obtain a solvent-substituted mugwort polymer fraction.
[482] (3) Solvent-substituted mugwort polymer fractions in a DEAE-cellophane A-800 column (Φ4.05 × 37.8 cm) equilibrated with 10 mM imidazole hydrochloride buffer (pH7.0) containing 10% ethanol and 50 mM sodium chloride. The column was washed with 1200 ml of the same buffer by addition, and eluted by a concentration gradient of sodium chloride from 0.1 M (1000 ml) to 2 M (1000 ml). The eluate was partitioned into 30 ml per piece. Among the elution fractions, fractions 13 to 33 were named mugwort leaf polymer fraction-I (YPS-I), and fractions No.69 to 78 were named mugwort leaf polymer fraction-II (YPS-II). Nos. 79 to 137 were named mugwort leaf polymer fraction-III (YPS-III). YPS-I, YPS-II and YPS-III were sufficiently dialyzed with distilled water and lyophilized. Each freeze-dried product was obtained with 530 mg, 420 mg, and 380 mg.
[483] (4) To fractionate YPS-III again, 200 mg of YPS-III was dissolved in 5 mM imidazole hydrochloride buffer (pH8.0) containing 50 ml of 4 M sodium chloride and equilibrated with the same buffer. -Added to cellulosic column (Φ 3.1 x 14.3 cm). After washing with 200 ml of the same buffer, eluted with 5 mM imidazole-hydrochloric acid buffer (pH8.0) containing 200 ml of 1 M sodium chloride, 200 ml of distilled water and 200 ml of ethanol, respectively.
[484] The eluate was partitioned into 10 ml each. Among the elution fractions, fractions 1 to 32 were named mugwort leaf polymer fraction-III-1 (YPS-III-1), and fractions No. 33 to 53 were fractionated mugwort leaf polymer fraction-III-2 (YPS-III- 2) and fractions No. 54 to 66 were named mugwort leaf polymer fraction-III-3 (YPS-III-3). YPS-III-1, YPS-III-2, and YPS-III-3 were sufficiently dialyzed with water and then lyophilized to obtain 20.11 mg, 32.59 mg, and 113.75 mg of the respective lyophilized products.
[485] (5) Fraction of mugwort extract prepared in Examples 21- (2) and 21- (3), YPS (sample ①), YPS-I (sample ②), YPS- in the same manner as in Example 1- (1) HGF production-inducing activity of II (sample ③) and YPS-III (sample 4) was measured. The results are shown in Table 80. As a result, fractions of these mugwort extracts exhibited HGF production inducing activity.
[486] sampleConcentration (µg / ml)Production of HGF (%) Sample ①0100 One214 10267 100215 Sample ②0100 One121 10113 100260 Sample ③0100 10129 100243 Sample ④0100 One232 10323 100272
[487] (However, the HGF production of the control group was 8.43 ng / ㎖)
[488] (6) Fraction of mugwort extract prepared in Example 21- (4) in the same manner as in Example 1- (1), YPS-III-1 (Sample ①), YPS-III-2 (Sample ②), YPS- HGF production inducing activity of III-3 (sample ③) was measured. The results are shown in Table 81. As a result, fractions of these mugwort extracts exhibited HGF production inducing activity.
[489] Addition amount (µg / mL)Production of HGF (%) Sample ①Sample ②Sample ③ 0100100100 One188290247 10181304217 100348295243
[490] (HGF production of the control group was 8.26 ng / ㎖)
[491] Example 22
[492] 30 g of fumeidan derived from Kagome kelp as described in Reference Example 1- (1) was dissolved in 12 L of distilled water at room temperature for 30 minutes. This suspension was centrifuged at 10000 xg for 40 minutes, and the supernatant was collected. These were filtered aseptically with a membrane filter (0.22 µm) (manufactured by Millipore) to obtain 21.4 g of a freeze-dried product. This was referred to as Takara Kelp Fucoidan Bf (hereinafter referred to as Fucoidan Bf).
[493] In the same manner as in Example 1- (1), HGF production inducing activity of Fucoidan Bf (sample ①) was measured. The results are shown in Table 82. However, the experiment was performed twice continuously and the average value was employ | adopted. As a result, fucoidan Bf showed HGF production inducing activity.
[494] Addition amount (µg / mL)Production of HGF (%) Sample ① 0100 One209 10333 100377
[495] (However, HGF production of the control group was 9.17 ng / ㎖)
[496] Example 23
[497] (1) 500 g of dried Kagome kelp was finely chopped, washed with 10 L of 80% ethanol, and then stirred in 25% of 10% ethanol containing 50 L of 1 mM potassium chloride at 25 ° C. for a diameter of 32 μm. Filtration was carried out with a stainless steel mesh to obtain about 45 L of filtrate. 34 L of this filtrate was heated at 80 degreeC for 3 hours, and it cooled to 50 degreeC. This was concentrated while maintaining the solution temperature at 50 ° C by ultrafiltration OMEGA cut (manufactured by Piltron) having a molecular weight of 10,000 cuts. Furthermore, desalination was carried out in 5 L of distilled water heated to 50 ° C, 200 ml of the distilled water was added, the flow path was washed twice and recovered to obtain 1.5 L of a concentrate. This was freeze-dried to obtain 8.2 g of F-rich fucoidan.
[498] (2) HGF production-inducing activity of F-rich fucoidan (sample ①) prepared in Example 23- (1) was measured in the same manner as in Example 1- (1). The results are shown in Table 83. As a result, F-rich showed HGF production inducing activity.
[499] Addition amount (µg / mL)Production of HGF (%) Sample ① 0100 One117 10186 100244
[500] (However, the HGF production of the control group was 9.60 ng / ㎖)
[501] Example 24
[502] NHDF cells (human normal skin fibroblasts) cultured in DMEM medium containing 10% fetal bovine serum were suspended in DMEM medium containing 10% fetal bovine serum to 1 × 10 ⁵ cells / ml, and 500 μl 48 The wells were placed in cell culture plates and incubated for 24 hours. Thereafter, the mixture was replaced with DMEM medium containing 1% fetal bovine serum, and 10 µg / ml or 100 µg / ml of minoxidil (manufactured by Wako Pure Chemical Industries, Ltd.) and a sample were added thereto. In addition, about the addition of F-rich fucoidan, the test was also performed about 1 microgram / ml addition of minoxidil. In addition, the division which adds only a sample without adding minoxidil was performed similarly. This medium was recovered and the amount of HGF in the medium was measured using a Quantikine Human Hepatocyte Growth Factor (HGF) ELISA Kit. As a sample, Fucoidan Bf prepared in Example 22, F-rich Fucoidan prepared in Example 23, and 7-12SFd-F prepared in Reference Example 2- (3) were added so as to have a final concentration of 1, 10, 100 µg / ml. It was. In addition, the fucoidan Bf was also tested for addition having a final concentration of 0.1 µg / ml. As a negative control, the same amount of distilled water as the sample was added. All experiments were performed three times in succession and the average value was adopted. The results are shown in Tables 84 to 86.
[503] In minoxidil-free, the amount of HGF in the medium increased concentration dependently of fucoidan Bf, F-rich fucoidan, 7-12SFd-F, and significantly increased than the unadded control. From this, it became clear that even when HGF mRNA such as minoxidil-free treatment was low, both the release of HGF on the cell surface and the synthesis of HGF could be promoted. In addition, when minoxidil was added, the HGF amount in the medium increased in a concentration-dependent manner of fucoidan Bf, F-rich fucoidan, and 7-12SFd-F, and significantly increased than the control group without addition. In addition, the increase in the amount of HGF when the mRNA was increased was very remarkable compared to the case where the amount of mRNA was small in the minoxidil treatment. From this, fucoidan Bf, F-rich fucoidan, and 7-12SFd-F promote the release and production of HGF to a lesser extent when mRNA is low, and when a large amount of mRNA is required because a large amount of mRNA is required. It has been clarified that this significantly promotes the release and production of HGF.
[504] HGF concentration (pg / ml) in NHDF / fucoidan Bf medium Fucoidan Bf (μg / ml)No minoxidilMinoxidil 10 μg / mlMinoxidil100 μg / ml 0N.D.126.04148.83 0.1300.20307.00478.90 One428.83448.17624.60 10791.93799.90794.20 100729.33709.97860.23
[505] N.D. Indicates below the detection limit.
[506] HGF Concentration (pg / ml) in NHDF / F-rich Fucoidan Medium F-rich fucoidan (µg / ml)No minoxidilMinoxidil 1 μg / mlMinoxidil 10 μg / mlMinoxidil100 μg / ml 0N.D.N.D.N.D.N.D. One132.75N.T.N.T.203.03 10316.90239.90310.20434.10 100369.33372.70372.70537.87
[507] N.D. Indicates below the detection limit. In addition, N.T. Indicates not to evaluate.
[508] HGF concentration (pg / ml) in NHDF / 7-12SFd-F medium 7-12SFd-F (μg / ml)No minoxidilMinoxidil 10 μg / mlMinoxidil100 μg / ml 0N.D.N.D.137.20 One228.73164.00378.30 10345.93376.07450.83 100439.67483.20820.10
[509] N.D. Indicates below the detection limit.
[510] Example 25
[511] A mouse (CDF1-based female, 7 weeks old, body weight: about 20 kg) was intraperitoneally administered with galactosamine (20 mg / mouse) and LPS (lipopolysaccharide: 0.03 µg / mouse), and a lethal model due to extreme hepatitis Was produced and the life-saving effect by the fucoidan of Reference Example 1- (1) was examined. Fucoidan was adjusted to 10% in distilled water and administered at a dose of 10 ml / kg body weight (1 g / kg as fucoidan) two times forced oral 1 hour before and 1 hour after co-administration of galactosamine and LPS. Distilled water was administered to the control group in the same manner.
[512] The survival rate after 72 hours from the start of the experiment was 1 of 8 cases in the control group and 7 of 8 cases in the fucoidan-administered group, and a significant life-saving effect was observed by fucoidan administration. Moreover, the improvement effect was also observed in the serum biochemical value in a survival example. The results are shown in Table 87.
[513] groupGPT (U / 1)GOT (U / 1)Total bilirubin (mg / dl) Control3856131.0 Fucoidan-administered group94 ± 22233 ± 530.3 ± 0.02
[514] Example 26
[515] (1) 500 g of kelp kelp are finely chopped and washed with 10 liters of 80% ethanol, followed by 2 minutes at 25 ° C. in a 40 cm inner container in 10% ethanol containing 50 liters of 1 mM potassium chloride for 1 day. A fucoidan solution was prepared by stirring at a speed of 120 revolutions per hour, extracting fucoidan, and extracting the extract with a stainless steel mesh having a mesh of 32 μm.
[516] 1 liter of palm oil solution prepared by dissolving 1 g of palm oil (Kaosha Co., Ltd.) in 1 liter of ethanol was added to 46 liters of the fucoidan solution while stirring, and again, 1 liter of glycerol was added to the lotion. It prepared.
[517] (2) Gelatin and perfume were added to the fucoidan solution prepared in Example 26- (1) so as to have a final concentration of 0.02% to obtain a lotion using gelatin. In addition, collagen was added in the same manner to obtain a lotion using collagen.
[518] Deposited biological materials
[519] (1) Name and destination of the depositary institution
[520] Institute of Commerce and Industry
[521] Japan Tsukubashi Higashi 1 Chome 1 Bar 3 Go (Postal Code 305)
[522] (2) deposited microorganisms
[523] (i) Alteromonas spp. SN-1009
[524] Won deposit date: February 13, 1996
[525] Date of Request for Escalation to International Deposit: November 15, 1996
[526] Accession number: FERM BP-5747
[527] (Ii) Flavoacterium spp. SA-0082
[528] Won deposit date: March 29, 1995
[529] Date of Request for Escalation to International Deposit: February 15, 1996
[530] Accession number: FERM BP-5402
[531] According to the present invention, a medicament effective for a disease requiring growth factor production containing as an active ingredient a substance that exhibits growth factor production-inducing activity is provided. The medicine has HGF production-inducing activity in vivo, h-IGF production-inducing activity, NGF and neurotrophic factor production-inducing activity, and the like. It is useful as a therapeutic or prophylactic agent for diseases.
[532] In addition, acid polysaccharides having sulfate-inducing action, sulfated polysaccharides such as fucoidan, dextran sodium sulfate, chondroitin sulfate high shark cartilage extract, their degradation products, acid oligosaccharides, acidic monosaccharides and salts thereof It is possible to produce food and drink, and by ingesting it as daily food and drink, it is possible to improve symptoms of diseases that require the production of growth factors. In addition, a feed having the same physiological function is provided.
[533] Therefore, a functional negative having an active ingredient selected from acidic polysaccharides, sulfated fucose-containing polysaccharides such as fucoidans, degradation products thereof, acidic oligosaccharides, acidic monosaccharides and salts thereof used in the present invention having HGF production inducing action. A food or functional food is a functional food or drink or a feed useful for maintaining homeostasis of a living body by its growth factor production inducing action.
[534] The present invention also provides a bio cosmetic for inducing HGF production, which is very useful for health care of the skin. Cancer metastasis inhibitors are also provided.
[535] In addition, a production inducer of growth factors is also provided, and the inducers are useful for studying the function of growth factors and for screening drugs for diseases related to the growth factors.

权利要求:
Claims (32)
[1" claim-type="Currently amended] It contains acid polysaccharides having an effect of inducing growth factor production, their degradation products, acid oligosaccharides, acidic monosaccharides, acidic sugar alcohols and salts thereof, except heparin and heparan sulfate as active ingredients. A therapeutic or preventive agent for a disease requiring growth factor production induction.
[2" claim-type="Currently amended] The therapeutic or prophylactic agent according to claim 1, wherein the acidic polysaccharide is a sulfated polysaccharide.
[3" claim-type="Currently amended] The therapeutic or prophylactic agent according to claim 2, wherein the sulfated polysaccharide is a sulfated polysaccharide derived from algae, a sulfated polysaccharide derived from an animal, a sulfated polysaccharide derived from a plant, a sulfated polysaccharide derived from a microorganism, or a sulfated polysaccharide derived from a fish.
[4" claim-type="Currently amended] The therapeutic or prophylactic agent according to claim 2, wherein the sulfated polysaccharide is a synthetic sulfated polysaccharide.
[5" claim-type="Currently amended] The therapeutic or prophylactic agent according to claim 2, wherein the sulfated polysaccharide is fucoidan.
[6" claim-type="Currently amended] The therapeutic or prophylactic agent according to claim 1, wherein the acidic monosaccharide is sulfated glucose, sulfated galactose, sulfated xylose, sulfated 2-deoxyglucose, sulfated dewose or sulfated mannose.
[7" claim-type="Currently amended] The method of claim 1, wherein the acidic oligosaccharides are sulfated maltose, sulfated lactose, sulfated sucrose, sulfated trehalose, sulfated lactulose, sulfated melibiose, sulfated cellobiose, sulfated isomaltose, sulfate Sulfurated touranose, sulfated palatinose, sulfated maltotriose, sulfated maltohexaose, sulfated maltoheptaose, sulfated dodecyl-maltohexaose, a compound represented by the following formula (I) and represented by the following formula (II) The therapeutic or prophylactic agent which is a sulfated oligosaccharide selected from the group consisting of:
[Formula I]
Wherein R is OH or OSO ₃ H
[Formula II]
(Wherein R is OH or OSO ₃ H).
[8" claim-type="Currently amended] The therapeutic or prophylactic agent according to any one of claims 1 to 7, wherein the growth factor is hepatocyte growth factor, insulin-like growth factor, or nerve growth factor.
[9" claim-type="Currently amended] The method according to any one of claims 1 to 8, further comprising an acid polysaccharide, a degradation product thereof, an acid oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, or a substance which synergistically increases the growth factor production-inducing action of these salts. Characterized in that the therapeutic or prophylactic agent.
[10" claim-type="Currently amended] 10. The compound according to claim 9, wherein the acid polysaccharide, the degradation product thereof, the acid oligosaccharide, the acid monosaccharide, the acid sugar alcohol, or a substance which synergistically increases the growth factor production-inducing action of these salts is a compound having cytokines, prostaglandins and cyclopentene rings. A therapeutic or preventive agent which is a substance selected from the group consisting of.
[11" claim-type="Currently amended] A growth factor production-inducing food, beverage or feed comprising an acid polysaccharide having a growth factor production-inducing action, a degradation product thereof, an acid oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, and a salt thereof.
[12" claim-type="Currently amended] The food, beverage or feed of Claim 11 wherein the acidic polysaccharide is a sulfated polysaccharide.
[13" claim-type="Currently amended] 13. The food, drink or feed according to claim 12, wherein the sulfated polysaccharide is a sulfated polysaccharide derived from algae, a sulfated polysaccharide derived from an animal, a sulfated polysaccharide derived from a plant, a sulfated polysaccharide derived from a microorganism, or a sulfated polysaccharide derived from a fish. .
[14" claim-type="Currently amended] 13. The food, drink or feed of Claim 12 wherein the sulfated polysaccharide is a synthetic sulfated polysaccharide.
[15" claim-type="Currently amended] 13. A food, beverage or feed according to claim 12, wherein the sulfated polysaccharide is fucoidan.
[16" claim-type="Currently amended] The food, beverage or feed according to claim 11, wherein the acidic monosaccharide is sulfated glucose, sulfated galactose, sulfated xylose, sulfated 2-deoxyglucose, sulfated dewose or sulfated mannose.
[17" claim-type="Currently amended] 12. The method of claim 11 wherein the acidic oligosaccharides are sulfated maltose, sulfated lactose, sulfated sucrose, sulfated trehalose, sulfated lactulose, sulfated melibiose, sulfated cellobiose, sulfated isomaltose, sulfate Sulfurated touranose, sulfated palatinose, sulfated maltotriose, sulfated maltohexaose, sulfated maltoheptaose, sulfated dodecyl-maltohexaose, a compound represented by the following formula (I) and represented by the following formula (II) Food, beverage or feed which is a sulfated oligosaccharide selected from the group consisting of:
[Formula I]
Wherein R is OH or OSO ₃ H
[Formula II]
(Wherein R is OH or OSO ₃ H).
[18" claim-type="Currently amended] 18. A food, beverage or feed according to any one of claims 11 to 17 for inducing hepatocyte growth factor production, insulin-like growth factor production or nerve growth factor production.
[19" claim-type="Currently amended] The method according to any one of claims 11 to 18, further comprising an acid polysaccharide, a degradation product thereof, an acid oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, or a substance which synergistically increases the growth factor production-inducing action of these salts. Characterized by food, beverage or feed.
[20" claim-type="Currently amended] 20. The compound according to claim 19, wherein the acid polysaccharide, its degradation product, acid oligosaccharide, acid monosaccharide, acid sugar alcohol, or a substance which synergistically increases the growth factor production-inducing action of these salts is a compound having cytokines, prostaglandins and cyclopentene rings. Food, drink or feed which is a substance selected from the group consisting of.
[21" claim-type="Currently amended] A growth factor production-derived cosmetic comprising an acid polysaccharide having a function of inducing growth factor production, a degradation product thereof, an acid oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, and a salt thereof.
[22" claim-type="Currently amended] The cosmetic according to claim 21, wherein the acidic polysaccharide is a sulfated polysaccharide.
[23" claim-type="Currently amended] The cosmetic according to claim 22, wherein the sulfated polysaccharide is a sulfated polysaccharide derived from algae, a sulfated polysaccharide derived from an animal, a sulfated polysaccharide derived from a plant, a sulfated polysaccharide derived from a microorganism, or a sulfated polysaccharide derived from a fish.
[24" claim-type="Currently amended] The cosmetic according to claim 22, wherein the sulfated polysaccharide is a synthetic sulfated polysaccharide.
[25" claim-type="Currently amended] The cosmetic according to claim 22, wherein the sulfated polysaccharide is fucoidan.
[26" claim-type="Currently amended] The cosmetic according to claim 21, wherein the acidic monosaccharide is sulfated glucose, sulfated galactose, sulfated xylose, sulfated 2-deoxyglucose, sulfated dewose or sulfated mannose.
[27" claim-type="Currently amended] The method of claim 21, wherein the acidic oligosaccharides are sulfated maltose, sulfated lactose, sulfated sucrose, sulfated trehalose, sulfated lactulose, sulfated melibiose, sulfated cellobiose, sulfated isomaltose, sulfate Sulfurated touranose, sulfated palatinose, sulfated maltotriose, sulfated maltohexaose, sulfated maltoheptaose, sulfated dodecyl-maltohexaose, a compound represented by the following formula (I) and represented by the following formula (II) Cosmetics which are sulfated oligosaccharides selected from the group consisting of:
[Formula I]
Wherein R is OH or OSO ₃ H
[Formula II]
(Wherein R is OH or OSO ₃ H).
[28" claim-type="Currently amended] The cosmetic according to any one of claims 21 to 27, for inducing hepatocyte proliferation factor production, for inducing insulin-like proliferation factor, or for inducing nerve growth factor.
[29" claim-type="Currently amended] 29. The method according to any one of claims 21 to 28, further comprising an acid polysaccharide, a degradation product thereof, an acid oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, or a substance which synergistically increases the growth factor production-inducing action of these salts. Cosmetics characterized by.
[30" claim-type="Currently amended] 30. The compound according to claim 29, wherein the acid polysaccharide, the degradation product thereof, the acid oligosaccharide, the acid monosaccharide, the acid sugar alcohol, or a substance which synergistically increases the growth factor production-inducing action of these salts is used in the compounds having cytokines, prostaglandins and cyclopentene rings. Cosmetics, the substance of choice.
[31" claim-type="Currently amended] The cosmetic according to any one of claims 21 to 30, which is a lotion, an emulsion, a cream, a pack, a bath solvent, a face wash, a bath soap, or a bath detergent.
[32" claim-type="Currently amended] A growth factor production regulator comprising an acidic polysaccharide, a degradation product thereof, an acidic oligosaccharide, an acidic monosaccharide, an acidic sugar alcohol, and one selected from the group consisting of these salts as an active ingredient.

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同族专利:

---

公开号 | 公开日

---

CN1192780C|2005-03-16|

---

JP4261071B2|2009-04-30|

---

KR100727339B1|2007-06-12|

---

CN1355703A|2002-06-26|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1999-04-15|Priority to JP10806799

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1999-04-15|Priority to JPJP-P-1999-00108067

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1999-04-15|Priority to JP10849999

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1999-04-15|Priority to JPJP-P-1999-00108499

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1999-04-22|Priority to JPJP-P-1999-00114542

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1999-04-22|Priority to JP11454299

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1999-05-10|Priority to JPJP-P-1999-00129163

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1999-05-10|Priority to JP12916399

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1999-05-21|Priority to JPJP-P-1999-00142343

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1999-05-21|Priority to JP14234399

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1999-06-02|Priority to JP15466299

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1999-06-02|Priority to JPJP-P-1999-00154662

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1999-07-14|Priority to JP20098299

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1999-07-14|Priority to JPJP-P-1999-00200982

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1999-09-28|Priority to JP27523199

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1999-09-28|Priority to JPJP-P-1999-00275231

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1999-12-28|Priority to JPJP-P-1999-00375606

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1999-12-28|Priority to JP37560699

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2000-03-31|Priority to JPJP-P-2000-00099941

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2000-03-31|Priority to JP2000099941

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2000-04-14|Application filed by 오미야 히사시, 다까라 슈조 가부시키가이샤

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2000-04-14|Priority to PCT/JP2000/002432

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2002-01-16|Publication of KR20020004997A

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2007-06-12|Application granted

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2007-06-12|Publication of KR100727339B1

优先权:

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申请号 | 申请日 | 专利标题

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JP10806799|1999-04-15|

---

JPJP-P-1999-00108067|1999-04-15|

---

JP10849999|1999-04-15|

---

JPJP-P-1999-00108499|1999-04-15|

---

JPJP-P-1999-00114542|1999-04-22|

---

JP11454299|1999-04-22|

---

JPJP-P-1999-00129163|1999-05-10|

---

JP12916399|1999-05-10|

---

JPJP-P-1999-00142343|1999-05-21|

---

JP14234399|1999-05-21|

---

JP15466299|1999-06-02|

---

JPJP-P-1999-00154662|1999-06-02|

---

JPJP-P-1999-00200982|1999-07-14|

---

JP20098299|1999-07-14|

---

JPJP-P-1999-00275231|1999-09-28|

---

JP27523199|1999-09-28|

---

JP37560699|1999-12-28|

---

JPJP-P-1999-00375606|1999-12-28|

---

JP2000099941|2000-03-31|

---

JPJP-P-2000-00099941|2000-03-31|

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PCT/JP2000/002432|WO2000062785A1|1999-04-15|2000-04-14|Remedies|