WO2007125823A1 - 免疫調節機能を有する多糖を主成分とする組成物 - Google Patents
免疫調節機能を有する多糖を主成分とする組成物 Download PDFInfo
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- WO2007125823A1 WO2007125823A1 PCT/JP2007/058591 JP2007058591W WO2007125823A1 WO 2007125823 A1 WO2007125823 A1 WO 2007125823A1 JP 2007058591 W JP2007058591 W JP 2007058591W WO 2007125823 A1 WO2007125823 A1 WO 2007125823A1
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- polysaccharide
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- cassis
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/02—Nasal agents, e.g. decongestants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
Definitions
- composition mainly composed of polysaccharide having immunomodulating function
- the present invention relates to a novel composition
- a novel composition comprising a polysaccharide as a main component, which can be obtained by limited partial decomposition of a polysaccharide fraction contained in a cassis juice with a specific enzyme.
- the present invention relates to an immunomodulator containing this composition and having an immunomodulatory effect such as an antitumor action or an antiallergic action, and further relates to a food or drink containing this composition.
- This composition has a very high immunomodulatory activity per unit amount compared to a polysaccharide derived from a conventional cassis juice, and at the same time has a very low viscosity, so that it is easy to handle in production.
- Background art
- innate immunity By the way, immune responses are roughly classified into innate immunity and acquired (adaptive) immunity.
- the innate immunity of the former is an immune system that is born in us, and phagocytes such as neutrophils, macrophages, and dendritic cells are representative of the cellular factors responsible for the biological defense mechanism. These cells use the help of humoral factors such as complement and lectin to recognize foreign substances such as microorganisms, and take them up and eliminate them quickly.
- humoral factors such as complement and lectin to recognize foreign substances such as microorganisms, and take them up and eliminate them quickly.
- innate immunity plays an important role not only in protecting early infection, but also in directing the subsequent induction of induction and differentiation of acquired (adapted) immunity, that is, the balance between cellular immunity and humoral immunity.
- Cellular immunity mainly consists of cytotoxic T cells (also known as killer T cells) and macrophages. Protects against staining, and humoral immunity controls the IgG1 and IgE antibody class switches in B cells, thereby infecting mainly microorganisms that grow outside the cell, such as protozoa, fungi, parasites, mycoplasma, pneumococci, and Escherichia coli. Work in defense.
- the origins of food and drink materials that adjust the balance of the immune system to a normal state include fungi represented by mushrooms, yeasts and lactic acid bacteria, seaweeds and herbs (see Non-Patent Documents 1 to 7). . These are collectively called biological response modifiers (BRM) from the meaning of regulatory action.
- BRM biological response modifiers
- Non-patent document 1 Medical mushrooms as a source of antitumor and immunomodulating polysaccharides. (Appl. Microbiol. Biotechnol., 60, 258-274 (2002))
- Non-patent document 2 Antitumor activity and immune response of Mekabu fucoidan extracte from Sporophyll of Undaria pinnatifida. (In Vivo, 17, 245-250 (2003))
- Non-Patent Document 3 Antitumor potential of a polysaccharide-rich substance from the fruit juice of Morinda citrifolia (Noni) on Sarcoma 180 ascites tumour in mice. (Phytother. Res., 17, 1158-1164 (2003))
- Non-Patent Document 4 A polysaccharide, extract from Gnfola frondosa, induces Th-1 domina nt responses in carcinoma-bearing BALB / c mice.
- Non-Patent Document 5 The anti-allergic effects of lactic acid bacteria are strain dependent a nd mediated by effects on both Thl / Th2 cytokine expression and balance. (Int. Arc h. Allergy Immunol., 135, 205-215 (2004) )
- Patent Document 6 In vitro ana in vivo anti-allergic activity of soy sauce. (Int. J. Mol. Med., 14, 879-884 (2004))
- Non-Patent Literature 7 Clinical effects of Lactobacillus acidophilus strain L-92 on perennial a llergic rhinitis: a double-blind, placebo-controlled study. (J. Dairy Sci., 88, 527-533 (2005))
- Non-Patent Document 8 Immunostimulatory Effects of a Polysaccharide-Rich Substance with Antitumor Activity Isolated from Black and urrant (Ribes nigrum L.). (Biosci. Biotechnol
- Patent Document 1 JP 2004-107660 A
- Cassis is commonly known as black currant, Japanese name is black currant, and is widely cultivated mainly in North America and recently in New Zealand. In particular, it is a very well-known material in the liquor confectionery industry.
- an object of the present invention is to provide a new material derived from cassis juice, which has an excellent antitumor effect similar to CAPS, etc., and is easy to handle with low viscosity, at a lower cost. .
- the present invention provides a novel substance derived from cassis fruit juice and having a physiological activity higher than that of CAPS, thereby providing a material having a superior physiological activity at lower cost.
- an object of the present invention is to provide an inexpensive and safe food and drink, particularly a healthy food and drink, using the above-described novel material having excellent physiological activity.
- CAPS is limited.
- a substance having an average molecular weight of about 10,000 to 40,000 produced by one partial enzymatic degradation exhibits an immunomodulatory effect higher than that of CAPS, and at the same time, its viscosity is lower than that of CAPS.
- the present invention has been completed.
- the present invention is as follows.
- the average molecular weight is in the range of 10,000 to 40,000
- composition according to [1] or [2], wherein the polysaccharide has an average molecular weight of about 20,000.
- composition power comprising a polysaccharide as a main component
- the production method according to [7] which is the composition according to any one of [1] to [5].
- An immunomodulator comprising the composition according to any one of [1] to [6], or the composition obtained by the production method according to [7] or [8].
- the immunomodulator according to [9] which has an antitumor action and an epilepsy or antiallergic action
- the immunomodulator according to [9] or [10] which has a hay fever suppressing action.
- the isolated CAPS and cassis juice itself have a higher immunomodulatory effect per unit amount and a lower viscosity, and are easily handled in the final product manufacturing process.
- This composition exhibits a higher immunomodulatory effect compared to conventional CAPS.
- immunomodulators and health foods and drinks that exhibit immunoregulatory effects such as antitumor action and antiallergic action using this composition as an active ingredient can also be provided.
- the composition of the present invention is a preparation from cassis juice that is eaten on a daily basis, it does not have any adverse effects on the human body even during long-term continuous use.
- composition comprising the polysaccharide of the present invention as a main component can be obtained by limited and partial enzymatic degradation of CAPS isolated from cassis juice or force cis juice itself.
- CAPS preparation from cassis juice can be performed according to the method described in JP-A-2004-107660 (Patent Document 1)
- Patent Document 8 various ionic substances are removed by passing the cassis juice (cassis supernatant of cassis puree) through cation exchange resin and anion exchange resin.
- the solution is passed through a C-18 reverse phase column to remove the polyphenol compound.
- the obtained flow-through fraction can be dialyzed with pure water and freeze-dried to obtain CAPS.
- CAPS is supplemented with ⁇ -galactosidase.
- the amount of ⁇ -galatatosidase added can be appropriately determined in consideration of the concentration of CAPS in the reaction solution, pH, reaction temperature, reaction time, the origin of galactosidase and the degree of purification. If the amount of the additive is too small, the reaction may take too much time S. In some cases, the enzyme may be deactivated in the process, and a degradation product having the desired molecular weight may not be obtained. . On the other hand, if the amount is too large, the reaction proceeds so rapidly that it becomes difficult to stop the reaction at an appropriate time, and it may increase the cost of manufacturing the product.
- the amount of ⁇ -galatatosidase added is 0.01% to 1% (w) when ⁇ -galatatosidase is a highly purified standard (reagent grade), for example. / v), preferably about 0.05% to 0.5% (w / v).
- the degree of purification of ⁇ -galatatosidase is inferior to that of a standard product.
- the addition amount (range) of the above / 3_galactosidase is merely an example and can be appropriately determined in consideration of the above conditions.
- the ⁇ -galactosidase used is most preferably derived from Aspergillus orvzae. However, as long as it is ⁇ -galatatosidase, other sources can be used as well. Examples of galactosidase derived from other sources include galactosidase derived from Kluvveromvces lactis; saccharomvces fragnis, Escherichia ⁇ 11, and the like.
- the peak on the low-molecular sugar (monosaccharide to oligosaccharide) side with a molecular weight of about 1,000 or less is not considered, and the peak on the polysaccharide side (MW> 1,000) is about 10,000 to
- the enzyme reaction is stopped when it is in the range of 40,000 to obtain a composition containing the target polysaccharide.
- a part of the reaction solution is sampled over time after the reaction is started, and the timing of the reaction stop can be monitored while analyzing by gel filtration by high-speed liquid chromatography (HPLC).
- a differential refraction detector is used as a high-performance liquid chromatography (HPLC) detector, and basically only sugars (monosaccharides, disaccharides, oligosaccharides, polysaccharides) are detected.
- the enzymatic reaction can be performed near the optimum temperature of the enzyme (eg, about 50 ° C.).
- the enzyme reaction can be stopped by boiling the enzyme for several minutes, for example 5 to 10 minutes, because the polysaccharide produced has high thermal stability.
- the composition of the present invention comprising a polysaccharide as a main component.
- the purification method is not particularly limited. For example, a small amount of insoluble precipitate generated by boiling can be removed by, for example, centrifugation. Furthermore, after removing the insoluble precipitate, low molecules (monosaccharide to oligosaccharide) having a molecular weight of 1,000 or less can be removed by, for example, subjecting to ultrafiltration having a molecular weight cut off of 1,000. If necessary, the composition of the present invention containing a polysaccharide as a main component can be obtained as a dried product by freeze-drying.
- the physicochemical properties of the polysaccharide contained in the composition comprising as a main component the polysaccharide of the present invention (hereinafter sometimes referred to as the present polysaccharide) finally obtained after purification are as follows (1) to ( As shown in 4).
- the average molecular weight is about 10,000 to 40,000, preferably about 15,000 to 25,000, and more preferably about 20,000. Average molecular weight is based on gel filtration analysis by HPLC.
- the polysaccharide contained in the composition of the present invention may further have the following physical chemical properties (5) and (6).
- the polysaccharide was positive for color reaction by phenol sulfate method and force rubazole sulfate method.
- the composition of the present invention containing the polysaccharide as a main component can also have the following physicochemical properties (7) to (9).
- Figure 1 shows a chart of the infrared absorption spectrum of the purified and lyophilized polysaccharide (MW> 1,000). Maximum absorption was observed around 1,000 cm— 1 .
- Fig. 2 shows an absorption spectrum chart of this polysaccharide (dissolved in pure water at a concentration of 80 mg / ml). Maximum absorption was seen at 194 nm.
- the content of the polysaccharide contained in the composition based on the polysaccharide of the present invention obtained by purification is, for example, in the range of 50 to 95% by mass, preferably in the range of 60 to 95% by mass, and more preferably. Is in the range of 80-95% by mass.
- composition of the present invention further contains a protein and polyphenolic compound in addition to the polysaccharide.
- the content of the protein and the polyphenol compound is in the range of 0 to 5% by mass and in the range of 5 to 45% by mass, respectively.
- the cassis juice used may be a puree containing all cassis fruit composition, or a so-called fruit juice from which insoluble components mainly derived from the skin and seeds have been removed in advance by a pretreatment such as centrifugation. good.
- the enzyme used is most preferably j3_galactosidase derived from AsDereillus orvzae as described above. However, other j3_galactosidase can be used as described above.
- the amount of ⁇ -galatatosidase added can be appropriately determined in consideration of the same points as in the limited and partial degradation of CAPS. For example, when ⁇ -galactosidase is a highly purified standard (reagent grade), for example, 0.01% to 1% (w / v), preferably about 0.05% to 0.5% (w / v) is appropriate. It is.
- the degree of purification of ⁇ -galatatosidase is inferior to that of the standard product, for example, 0.2% to 2% (w / v) is appropriate.
- the enzyme treatment / deactivation * sterilization conditions are as mild as possible.
- enzyme treatment can be performed at around 40 ° C, which is close to physiological conditions.
- the reaction temperature is preferably lower than the enzymatic decomposition of isolated or partially purified CAPS.
- a part of the reaction solution is sampled over time after the enzyme reaction is started, and the reaction stop time is determined by analyzing it by gel filtration using high-performance liquid chromatography (HPLC) using a differential refraction detector as the detector. can do.
- HPLC high-performance liquid chromatography
- the peak on the low-molecular-weight saccharide (monosaccharide to oligosaccharide) side with a molecular weight of about 1,000 or less is not taken into account, and when the peak on the polysaccharide side (MW> 1,000) shows an average molecular weight of about 20,000, stop. Since the pH of cassis is very low at 2.9, the enzyme can be deactivated by holding it at around 70 ° C for several minutes. This operation can also be sterilized.
- a composition containing the above polysaccharide as a main component can be obtained from the cassis juice.
- the viscosity of the cassis juice decreases to about 1/5 depending on the degree of degradation before and after the enzyme treatment. Therefore, the product has properties that are very easy to handle.
- the content of the polysaccharide contained in the composition based on the polysaccharide of the present invention obtained from the cassis fruit juice is in the range of 50 to 95% by mass.
- composition of the present invention obtained from cassis juice further contains a protein and a phenolic compound in addition to the polysaccharide.
- the content of protein and polyphenol compound is in the range of 0-5% by mass and in the range of 5-45% by mass, respectively.
- the present invention also includes an immunomodulator and a food or drink containing the composition comprising the polysaccharide of the present invention as a main component.
- a composition containing a polysaccharide component newly produced from a polysaccharide (main component: CAPS) contained in a cassis fruit exhibits an immunomodulatory effect accompanied by an antitumor action, an antiallergic action and the like.
- the above composition of the present invention has a hay fever suppressing effect and can be used as an immunomodulator. It is specifically shown in the examples described later that the above composition of the present invention has an effect for suppressing various symptoms of human cedar pollinosis.
- the immunomodulator is obtained by mixing a composition containing a polysaccharide derived from cassis fruit juice (CAPS) with various ingredients, and removing water by a spray drying method or a freeze drying method.
- the various materials that can be used include sweeteners, colorants, storage stabilizers, antioxidants, acidulants, flavorings, bitterings, spices, and seasonings.
- excipients that can be used include lactose, crystalline cellulose, starch (dextrin), sucrose fatty acid ester, glycerin fatty acid ester and the like.
- the immunomodulator of the present invention is generally administered orally, and the dosage is determined according to a doctor's prescription based on the patient's symptoms, age, weight, and the like.
- the general range of dosage is, for example, 0. lg to:! OOg.
- Administration can be done in one or more divided doses.
- food and drink containing the above-described polysaccharide-based composition of the present invention can also be used for regulating immunity.
- the food or drink of the present invention is used for regulating immunity, and can also be a food or drink with "indication to be used for regulating immunity".
- “indication” includes, for example, not only indications on food and beverage containers and instruction manuals, but also advertising texts and audio advertisements on food and beverages posted on paper media and on the web. .
- a food or drink with a label means not only a food or drink with a label on a food or drink container or an instruction manual, but also a container or an instruction manual without a label. This includes cases where the same indication is made by advertising texts or voice advertisements about the food and beverages posted on the paper and on the web. For example, “For people with weak constitution”, “For nutrition and toughness”, “For constitution improvement”, “For people who tend to catch a cold”, “For people who are sensitive to the season”, “For people who are sensitive to early spring” ”,“ For people with sensitive skin ”,“ at the time of the examination ”,“ for maintaining physical condition ”, etc.
- the food or drink of the present invention is obtained by blending the composition of the present invention having the above-mentioned immunomodulating effect into a food or drink according to a standard method, and the subject to consume it is limited to humans. It is also applicable to pets such as naguineu and cats and all animals. That is, in the present invention, the food and drink includes animal feed.
- the food and drink of the present invention is intended for immunomodulation including the above-described improvement of immunity and enhancement. It is prepared by mixing active ingredients prepared from cassis fruit.
- active ingredients prepared from cassis fruit.
- As the form of food all forms such as solid food, semi-fluid food such as cream, gel food, beverage and the like are possible. Alternatively, it may be in the form of powder, granule, capsule, tablet, liquid, etc.
- the various components to be blended in the food and drink together with the active ingredients prepared from the cassis fruit can be any of various commonly used components with no particular restrictions.
- examples of such components include glucose, fructose, sucrose, maltose, sonolebithonole, ratatoose, citrate, tartaric acid, malic acid, succinic acid, lactic acid, casein, gelatin, pectin, agar, amino acids, dyes , Fragrances, preservatives and the like.
- Specific examples of the food according to the present invention include soft drinks, juices, jams, confectionery, dairy products and the like.
- the content of the active ingredient prepared from the fruit of the cassis should be in the range of 0.01 to 100 mg / g. There is no problem even if it is added in a larger amount than this range.
- Example 1 Preparation of a composition of the present invention from isolated CAPS
- CAPS was basically isolated according to Patent Document 1 and Non-Patent Document 8.
- the preparation method is described briefly below.
- Cassis juice obtained by centrifuging Cassis' puree is packed with equal amounts of cation exchange resin (Amberlite IR 120B H AG [manufactured by Onoregano]) and anion exchange resin (IRA 410000 H AG [manufactured by Organo]).
- the mixed bed column was passed at a flow rate of SV 4, and the flow-through fraction was collected.
- the obtained fraction was passed through 35 ml of S mark — Pak C18 Vac (manufactured by Millipore) to adsorb the polyphenol compound to the solid phase resin, and the non-adsorbed fraction was collected.
- CAPS was obtained by freeze-drying.
- Patent Document 1 the ability to fractionate CAPS into polysaccharide A and polysaccharide B by ethanol precipitation
- CAPS is a mixture of polysaccharide A and polysaccharide B.
- PBS phosphate buffered saline
- ⁇ 7.4 phosphate buffered saline
- Dissolved in 1 Adjust to ⁇ 5.5 with hydrochloric acid, and add ⁇ -galactosidase (Sigma, As ⁇ illus orvzae) was added at 0.1% w / v (8 units / ml), incubated at 50 ° C, sampled over time, and boiled for 10 minutes to inactivate the enzyme.
- the centrifugal supernatant of each sample was subjected to gel filtration analysis by HPLC, and the sampnore of the composition containing the polysaccharide of the present invention in which the average molecular weight of the polysaccharide fraction (MW> 1,000) was about 20,000 was identified.
- the analytical column is Shodex OHpak SB_804 (exclusion limit: MW 1, 000,000), the equilibration buffer is PBS, the analysis flow rate is 1 ml / min, and the detector is a differential refractometer (RI) D) was used.
- Molecular weight markers include T-2000 (MW 2,000,000), T-500 (MW 473, 000), T-70 (MW 67,200), T-40 (MW 43,000), T-10 (MW 10,000) [more Manufactured by Falmasia Co., Ltd.], maltohexaose (MW 991) sucrose (MW 342), glucose (MW 180) were used.
- the behavior of the active fraction was examined by changing the concentration of ethanol added by dissolving this polysaccharide in PBS to 4 mg / ml.
- ethanol was added at various concentrations, and the supernatant fraction and the precipitate fraction were separated by centrifugation (15,000 rpm, 10 minutes), and PBS was added to the precipitate fraction and suspended in the original volume. .
- PBS was added in the same manner to the original volume.
- the TNF- ⁇ (a kind of cytodynamic force released from macrophages) induction activity of the supernatant fraction and the precipitate fraction at each ethanol concentration was measured.
- each fraction was subjected to gel filtration analysis by HPLC.
- the method for measuring TNF- ⁇ -inducing activity is as follows.
- the mouse macrophage-like cell line RAW 264 was used (1 X 10 5 cells / ml, 100 ⁇ 1, 37 ° C, grown in 5% CO.
- Fig. 3 shows the chromatograms of the analytical samples after 0 (undigested; boiled for 10 minutes after addition of a predetermined amount of previously inactivated enzyme), 3, 8, and 35 hours together with a calibration curve. After 35 hours Sampunore is completely digested by enzymes. The average molecular weights of the produced polysaccharides after 3, 8, and 35 hours were 55,000, 19,000, and 2400, respectively. That is, polysaccharides were produced in the 8-hour digested sample.
- the fraction obtained by removing low-molecular-weight (monosaccharide to oligosaccharide) molecules with a molecular weight of 1,000 or less from this polysaccharide-containing product consists only of rhamnose, mannose, arabinose, galactose, xylose, and glucose as neutral sugars.
- the constituent molar ratio was 18: 3: 19: 30: 1: 29.
- Fig. 4 shows the behavior of the active fraction in an ethanol aqueous solution. Ethanol concentration is 30%
- the TNF-stimulation activity present in the supernatant decreased rapidly.
- the ethanol concentration reached 40% or higher the activity present in the supernatant almost disappeared.
- the polysaccharide S present in the supernatant was almost undetectable, and the activity was not completely transferred to the precipitate fraction.
- Example 2 Preparation of a composition of the present invention from Cassis' puree (juice)
- SVZ Cassis' puree
- Sumiratato L ⁇ -galactosidase-based food enzyme preparation, derived from Aspergillus orvzae, manufactured by Shin Nippon Chemical Industry Co., Ltd.
- the enzyme was inactivated by sampling a small amount over time and boiling for 10 minutes. Centrifugation (15,000 rpm, 10 minutes) supernatant was subjected to HPLC analysis and viscosity measurement.
- the analysis conditions for gel filtration are the same as in Example 1.
- the viscometer used was VISCOMATE (model VM-1G-L, manufactured by Yamaichi Electronics Co., Ltd.).
- Fig. 5 shows a gel filtration chromatogram of a case of reacting at 1% (w / v) at 40 ° C for 6 hours and an untreated puree.
- Example 2 As a result of Example 2, a composition comprising the polysaccharide of the present invention as a main component can be obtained from a puree that does not impair the flavor due to extremely mild enzyme reaction conditions and relatively mild deactivation. It has become possible. [0061]
- Example 3 Measurement of site force-in-inducing activity
- Peritoneal macrophages were prepared according to a conventional method. Specifically, thioglycolate medium was injected intraperitoneally into an ICR mouse, and after 4 days, peritoneal macrophages were collected and finally dispensed into a 96-well plate at 1 X 10 6 cells / ml. Used for testing.
- TNF-s present in the macrophage culture medium after overnight culture was measured using an ELISA quantification kit (Quantikine mouse TNF-s, manufactured by R & D Systems) according to the attached manual.
- composition of the present invention obtained by treating CAPS with ⁇ -galactosidase has a site-force-in inducing activity that is nearly 10 times higher in CA than CAPS.
- mice Female ICR mice (SPF) purchased at 4 weeks of age from Japan SLC were preliminarily raised for 8 days and used for experiments. Mice were raised in the SPF animal room (lighting time 8-18 hours, ventilation rate 18 times / hour) at room temperature 24 ⁇ 3 ° C and relative humidity 55 ⁇ 15% throughout the preliminary and experimental periods. The mice were 5 animals / cage, and sterilized distilled water was freely given in a water supply bottle, and solid feed (MF, oriental yeast) was freely given in a mouse feeder. In addition, the individual identification of mice was performed using a dye (picric acid solution) coating method.
- day 14 The day when the tumor cells were transplanted was defined as day 0, and administration (10 mice per group) was performed by oral gavage once a day from day _6 to day 14. On the last day (day 14), the mouse was dissected to remove the tumor, and then the weight was measured with an electronic balance.
- composition having the polysaccharide as a main component obtained in Example 1 was measured for its antiallergic action.
- the test system used was a mouse allergy model induced with ovalbumin.
- the experimental animals were 8-8 male mice purchased at 6 weeks of age from Nippon Chisuriba Corporation.
- mice were preliminarily raised for one day. Mice were raised in a SPF barrier breeding room (lighting time 7-19 hours, ventilation rate 18 times / hour) at room temperature 24 ⁇ 3 ° C and relative humidity 55 ⁇ 15% throughout the preliminary breeding period and experimental period. There were 5 mice / cage, and sterilized distilled water was freely given in a water bottle, and mouse chow (MF, Oriental Yeast Co., Ltd.) was freely given in a feeder. For identification of mice, picric acid was applied to the hair.
- the preparation of dosing sampnore was according to the method described in Example 1.
- the average molecular weights of the administration samples used in this example were MW 59,000, MW 24,000, and MM 3,500, respectively.
- Dilute the partial digestion reaction solution 10 times for each sample, including undigested CAPS (Polysaccharide concentration: 400 ⁇ g / ml) was used as an administration sample.
- the administration method was gavage (10 ml / kg).
- Cyclophosphamide (30 mg / kg, 3 mg / ml, 0.5% CMC suspension) was used as a positive control.
- the administration sample was forcibly orally administered every day from day 15 to day 21 (the first immunization day was day 0) using an oral mouse for mice.
- the administration volume was 0.1 ml per 10 g body weight of mice.
- Ovalbumin (OVA, manufactured by Seikagaku Corporation) was used as a sensitizing antigen.
- An appropriate amount of OVA for primary immunization was weighed and dissolved in physiological saline to prepare a 66 ug / ml solution. 10 ml of this OVA solution and aluminum hydroxide gel ( ⁇ 1 ( ⁇ ), 13 mg / ml, manufactured by Sigma)
- 0.3 ml of the antigen for primary immunization was intraperitoneally administered twice a day 0 and day 4 to a 6-week-old mouse.
- nasal sensitization was performed by placing the antigen solution for secondary immunization into a centrifuge tube and immersing the mouse's nose for 3 seconds. Nasal sensitization was performed 3 times a day during days 9-13.
- Body weight was measured with a scale on day 0, day 7, day 14 and day 22. General symptoms were observed daily from day 0 to day 22.
- the plasma was analyzed by measuring the OVA-specific IgE level using the plasma after centrifugation of blood collected from the eye vein on day 0, day 14, day 18 and day 22 respectively.
- OVA-specific IgE levels were determined by storing the plasma of individuals with high VA-specific IgE levels at ⁇ 80 ° C, using it as a positive control, creating a dilution series and drawing a standard curve, and relative activity against it. Expressed in
- FIG. 8 shows the measurement results of the OVA-specific IgE level in blood (plasma) collected on the last day (day 22). Only CAPS degradation products with an average molecular weight of about 20,000, ie, this polysaccharide, showed a tendency to suppress IgE elevation.
- test is a double-blind comparative study with the placebo group as a control compared to the test product group, and the test method is described below.
- Example 2 Cassis puree was subjected to enzyme treatment (Sumiratat L 10,000 U / g was added at a final concentration of 0.9% [w / v] and reacted at 42 ° C for 5 hours). Thereafter, the enzyme was inactivated at 70 ° C for 10 minutes. Next, the supernatant was collected by centrifugation, dextrin was added to the resulting supernatant containing the polysaccharide of the present invention (CAPS) as a main component, and freeze-dried.
- enzyme treatment Sudiratat L 10,000 U / g was added at a final concentration of 0.9% [w / v] and reacted at 42 ° C for 5 hours. Thereafter, the enzyme was inactivated at 70 ° C for 10 minutes. Next, the supernatant was collected by centrifugation, dextrin was added to the resulting supernatant containing the polysaccharide of the present invention (CAPS) as a main component, and freeze-dried.
- this dried product (dextrin content 49.2% [w / w]) is pulverized, and the powdered fragrance Cassismicron M-10759 (manufactured by Takasago International Corporation) is added and mixed at 0.6% / w].
- the tableting powder was obtained. Tableting was performed using this tableting powder to obtain 2 g of a tabletable tablet per tablet.
- Specific IgE antibody titers were measured before taking the test article, 4 weeks and 8 weeks later, and the average antibody titer was compared between the test article group and the placebo group. There was no difference in the antibody titer between the test product group and the placebo group.
- composition comprising a polysaccharide derived from blackcurrant juice (CAPS) of the present invention as a main component has an effect of improving or preventing "itching of the eyes” that is a main symptom of cedar pollinosis, and is effective in reducing nasal symptoms.
- CAPS blackcurrant juice
- Yo it was suggested that the effect can be expected to alleviate various symptoms of Japanese cedar pollinosis with allergic symptoms. In addition, it can be taken safely for a long time because there are no side effects.
- the novel composition of the present invention comprising as a main component a polysaccharide that can be obtained by limited partial decomposition of the polysaccharide fraction contained in the cassis fruit juice of the present invention with a specific enzyme is a food and drink product and a pharmaceutical product. It is useful in the field of
- FIG. 1 shows an infrared absorption spectrum of the composition of the present invention.
- FIG. 2 shows an absorption spectrum of the composition of the present invention.
- FIG. 3 A gel filtration chromatogram of a polysaccharide contained in the composition of the present invention is shown together with a calibration curve.
- FIG. 4 shows the activity behavior of polysaccharides contained in the composition of the present invention in an aqueous ethanol solution.
- FIG. 5 shows gel filtration chromatograms of cassis puree before and after enzyme treatment in Example 2.
- FIG. 6 shows the measurement results of site force-in-inducing activity in Example 3.
- FIG. 7 shows a mouse antitumor effect of the composition of the present invention in Example 4.
- FIG. 8 shows the antiallergic effect of the composition of the present invention in Example 5.
- FIG. 9 shows changes in body weight of mice administered with the composition of the present invention in the antiallergic test in Example 5.
- FIG. 10 shows changes in doctor's nasal cavity swelling score before and after intake of test food in the pollinosis human intervention study in Example 6.
- FIG. 11 shows the change in QOL score for each symptom before and after intake of test food in the pollinosis human intervention study in Example 6.
Abstract
Description
Claims
Priority Applications (6)
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US12/226,671 US7879370B2 (en) | 2006-04-26 | 2007-04-04 | Composition of which chief ingredient is polysaccharides having an immunoregulatory function |
JP2008513171A JPWO2007125823A1 (ja) | 2006-04-26 | 2007-04-20 | 免疫調節機能を有する多糖を主成分とする組成物 |
EP07742027.1A EP2017301B1 (en) | 2006-04-26 | 2007-04-20 | Composition of which chief ingredient is polysaccharides having an immunoregulatory function |
PL07742027T PL2017301T3 (pl) | 2006-04-26 | 2007-04-20 | Kompozycja, której głównym składnikiem są polisacharydy mające funkcję immunoregulatorową |
AU2007244497A AU2007244497B2 (en) | 2006-04-26 | 2007-04-20 | Composition of which chief ingredient is polysaccharides having an immunoregulatory function |
NZ572501A NZ572501A (en) | 2006-04-26 | 2007-04-20 | Composition of which chief ingredient is polysaccharides having an immunoregulatory function |
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JP2006-121492 | 2006-04-26 | ||
JP2006121492 | 2006-04-26 | ||
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JP2006286171 | 2006-10-20 |
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US (1) | US7879370B2 (ja) |
EP (1) | EP2017301B1 (ja) |
JP (2) | JPWO2007125823A1 (ja) |
AU (1) | AU2007244497B2 (ja) |
NZ (1) | NZ572501A (ja) |
PL (1) | PL2017301T3 (ja) |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2011201781A (ja) * | 2010-03-24 | 2011-10-13 | Morishita Jintan Co Ltd | 抗アレルギー剤 |
WO2012020840A1 (ja) * | 2010-08-13 | 2012-02-16 | キリンホールディングス株式会社 | 肌質改善用組成物 |
US20130280357A1 (en) * | 2011-01-06 | 2013-10-24 | Johannes Coy | Chocolate mass |
JP2015065927A (ja) * | 2013-09-30 | 2015-04-13 | キリン株式会社 | カシス果実由来の多糖含有組成物の製造方法 |
CN113980152A (zh) * | 2021-12-01 | 2022-01-28 | 吉林农业大学 | 一种桦褐孔菌均一多糖及其制备方法和应用 |
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- 2007-04-20 WO PCT/JP2007/058591 patent/WO2007125823A1/ja active Application Filing
- 2007-04-20 AU AU2007244497A patent/AU2007244497B2/en not_active Ceased
- 2007-04-20 JP JP2008513171A patent/JPWO2007125823A1/ja active Pending
- 2007-04-20 EP EP07742027.1A patent/EP2017301B1/en not_active Not-in-force
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Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2011201781A (ja) * | 2010-03-24 | 2011-10-13 | Morishita Jintan Co Ltd | 抗アレルギー剤 |
WO2012020840A1 (ja) * | 2010-08-13 | 2012-02-16 | キリンホールディングス株式会社 | 肌質改善用組成物 |
JPWO2012020840A1 (ja) * | 2010-08-13 | 2013-10-28 | キリンホールディングス株式会社 | 肌質改善用組成物 |
AU2011290175B2 (en) * | 2010-08-13 | 2014-08-07 | Kirin Holdings Kabushiki Kaisha | Composition for improving skin quality |
JP2016084356A (ja) * | 2010-08-13 | 2016-05-19 | キリンホールディングス株式会社 | 肌質改善用組成物 |
US20130280357A1 (en) * | 2011-01-06 | 2013-10-24 | Johannes Coy | Chocolate mass |
US10694761B2 (en) * | 2011-01-06 | 2020-06-30 | Johannes Coy | Chocolate mass |
JP2015065927A (ja) * | 2013-09-30 | 2015-04-13 | キリン株式会社 | カシス果実由来の多糖含有組成物の製造方法 |
CN113980152A (zh) * | 2021-12-01 | 2022-01-28 | 吉林农业大学 | 一种桦褐孔菌均一多糖及其制备方法和应用 |
CN113980152B (zh) * | 2021-12-01 | 2022-09-13 | 吉林农业大学 | 一种桦褐孔菌均一多糖及其制备方法和应用 |
Also Published As
Publication number | Publication date |
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AU2007244497B2 (en) | 2011-05-12 |
US7879370B2 (en) | 2011-02-01 |
JPWO2007125823A1 (ja) | 2009-09-10 |
AU2007244497A1 (en) | 2007-11-08 |
JP5632054B2 (ja) | 2014-11-26 |
NZ572501A (en) | 2010-12-24 |
EP2017301B1 (en) | 2013-10-30 |
US20090220531A1 (en) | 2009-09-03 |
PL2017301T3 (pl) | 2014-04-30 |
EP2017301A4 (en) | 2011-08-10 |
EP2017301A1 (en) | 2009-01-21 |
JP2014015616A (ja) | 2014-01-30 |
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