JPS6153328B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION Technical Field The present invention relates to a novel anti-inflammatory polysaccharide and a method for producing the same. More specifically, the present invention relates to an anti-inflammatory polysaccharide isolated and purified from a hot water extract of Melia azadirachta bark, and a method for producing the same.
The polysaccharide of the present invention is useful as a low toxicity anti-inflammatory agent. Prior Art It has been known that Melia azadirachta extracts have various pharmacological actions. That is, a method for obtaining skin cosmetics by extracting the bark, leaves, flowers, fruits, branches, root bark, or resin of Melia azadirachta with water or a hydrophilic solvent or by pulverizing it (Japanese Patent Publication No. 52-28853) , 52-28854 and 53-
10125), extracting the above Melia azadirachta raw material with a hydrophilic solvent and/or hot water to obtain antibacterial activity,
A method for obtaining ingredients that improve gastrointestinal and liver function (Japanese Patent Publication No. 53-10124) and a method for extracting the above-mentioned Melia azadirachta raw materials with a hydrophobic solvent to obtain ingredients effective for the treatment of skin diseases and rheumatism (Japanese Patent Publication No. 53-10124) −13689) has been reported. As a result of repeated research on Melia azadirachta bark components, the present inventors isolated a polysaccharide consisting of glucose, arabinose, and fucose as a pharmacologically active component of Melia azadirachta bark hot water extract. This polysaccharide is a new compound that has not been described in any literature and has excellent anti-inflammatory effects. OBJECTS OF THE INVENTION It is therefore an object of the present invention to provide novel anti-inflammatory polysaccharides obtained from Melia azadirachta bark. As described below, the polysaccharide of the present invention exhibits a strong inhibitory effect on carrageenan edema. A further object of the present invention is to provide a method for producing the above polysaccharide. According to the method of the present invention, the polysaccharide is produced by treating a hot water extract of Melia azadirachta bark with a molecular sieve, particularly gel filtration, and collecting a compound with a molecular weight of about 8,000. DETAILED DESCRIPTION OF THE INVENTION The present invention consists first of all of an anti-inflammatory polysaccharide obtained from the bark of Melia azadirachta and having the following properties. (a) Color and shape White or slightly brown powder (b) Solubility Soluble in water, insoluble in organic solvents such as methanol, ethyl acetate, and benzene. (c) Sugar composition Consisting of glucose, arabinose and fucose in a composition ratio of 6:1:1. (d) Molecular weight approximately 8000 (e) Specific rotation [α] ²² _D : +16.7° (c = 0.5, H ₂ O) (f) Ultraviolet absorption spectrum No absorption maximum was observed when measured in an aqueous solution, and the terminal absorption was shows. The second aspect of the present invention comprises a method for producing the above-mentioned anti-inflammatory polysaccharide, which comprises subjecting a hot water extract of Melia azadirachta bark to molecular sieving to collect a compound having a molecular weight of about 8,000. Thirdly, the present invention provides the above-mentioned molecular sieve treatment to first reduce the molecular weight cut-off from about 1×10 ³ to 1×10 ⁵ to 1×10 ³ to 2×
10 ⁵ molecular sieve, and of the three fractions of the polysaccharide obtained, the third eluting fraction is collected, and this is further purified using an alkylated dextran gel. Consists of a method for producing an anti-inflammatory polysaccharide. Fourthly, the present invention comprises a method for producing the anti-inflammatory polysaccharide, wherein the molecular sieving treatment is gel filtration, and the molecular sieve agent is a gel filtration agent. Fifth, the present invention comprises a method for producing the anti-inflammatory polysaccharide, wherein the gelling agent is dextran gel, polyacrylamide gel, hydrophilic polyvinyl gel, or porous glass beads. When carrying out the method of the present invention, Melia
A hot water extract of Azadirachta bark is subjected to molecular sieve treatment, particularly gel filtration, to collect a compound having a molecular weight of about 8000. Melia azadirachta, which is a raw material plant for the method of the present invention, is a woody plant that grows naturally in tropical regions and reaches a height of 10 m or more. In the method of the present invention, a hot water extract of Melia azadirachta bark is used as a raw material. The extraction treatment of Melia azadirachta bark with hot water is carried out according to conventional methods. That is, it is carried out by adding hot water to shredded Melia azadirachta bark, or by adding water to Melia azadirachta bark and heating the mixture to boiling. Heating can be carried out in a boiling water bath or over an open fire. The extraction time is appropriately determined depending on the quality of the raw materials, etc., but is usually 1 to 48 hours. After the extraction is completed, the extract mixture is filtered to obtain an extract. Since the Melia azadirachta bark hot water extract thus obtained contains a large amount of impurities, it is necessary to purify the extract by alcohol precipitation or dialysis membrane method before applying it to the gel filtration process of the present invention. is desirable. For example, when purifying by alcohol precipitation, an alcohol such as methanol or ethanol is added to the above extract, and the resulting precipitate is collected by a conventional method, for example, by centrifugation. When purifying by dialysis membrane method, put the extract into a dialysis membrane,
When using a membrane that fractionates substances with a molecular weight of 20,000 or more by dialysis by soaking in water, the external dialysis solution is
When using a membrane that fractionates 14,000 or more substances, the dialyzed fluid is concentrated to dryness or freeze-dried to obtain an extract. As the dialysis membrane, for example, Spectra Pore (product of Spectrum Medical Industries) and Visking Tube (product of Union Carbide) are used. The extract obtained by purification by alcohol precipitation or dialysis membrane method is dissolved in water to obtain a hot water extract of Melia azadirachta bark, which is the raw material for the method of the present invention. Furthermore, prior to the hot water extraction treatment of the Melia azadirachta bark, it is also desirable to pre-extract the Melia azadirachta bark with an organic solvent and/or water at room temperature to remove unnecessary components in advance. Solvents used for extraction pretreatment include polar organic solvents such as methanol, ethanol, propanol, pyridine, and acetone, and nonpolar organic solvents such as benzene, toluene, xylene, n-hexane, chloroform, carbon tetrachloride, and ethyl acetate. can be given. In the gel filtration step in the method of the present invention, the hot water extract of Melia azadirachta bark is applied to a column packed with a gel filter agent, and a polysaccharide having a molecular weight of about 8000 is collected from the eluted polysaccharide fraction by a conventional method. It will be implemented. Examples of the gelling agent used in the above step include dextran gel, polyacrylamide gel, polyvinyl polymer gel, and porous glass beads. These include, for example, Cephadex G-50, G-75, G-100 (products of Pharmacia, Sweden), Biogel P-10 to P-30.
(BioRad product, USA), Toyopearl HW-
50, HW-55 (Toyo Soda Kogyo Products, Japan), CPG
It is commercially available under product names such as -10 (Electro Nucleonics, USA). Furthermore, in the method of the present invention, the Melia azadirachta bark hot water extract has a molecular weight cut-off of about 1×10 ³ to 1×10 ⁵ to 1
A column filled with ×10 ³ to 2 × 10 ⁵ gelatinizer was applied to the column and eluted with distilled water. Of the three polysaccharide fractions obtained, the third eluting fraction was collected and further alkylated. A column packed with dextran gel, such as Sephadex LH-20, is collected, and a fraction eluted with distilled water is collected and evaporated to dryness or freeze-dried to obtain the desired anti-inflammatory polysaccharide. However, if the hot water extract of Melia azadirachta bark is treated with a dialysis membrane with a molecular weight cut-off of 20,000 or more and the dialyzed liquid is used as a raw material in the method of the present invention, it should be gel-filtered with the gel-filtering agent mentioned above. No particular process is required. The polysaccharide thus obtained has the following properties. (a) Color and shape White or slightly brown powder (b) Solubility Soluble in water, insoluble in organic solvents such as methanol, ethyl acetate, and benzene. (c) Sugar composition Consisting of glucose, arabinose and fucose in a composition ratio of 6:1:1. (d) Molecular weight: Approximately 8000 This molecular weight can be determined by the Park-Johnson method [Park &Johnson; J, Biol.Chem. 181 , 149
(1949)] by quantifying reducing sugars. (e) Specific rotation [α] ²² _D : +16.7° (c=0.5, H ₂ O) (f) Ultraviolet absorption spectrum When measured in an aqueous solution, it does not show an absorption maximum, but shows terminal absorption. Next, the method for producing a polysaccharide of the present invention will be explained in more detail with reference to Reference Examples and Examples. Reference Example Production of hot water extract of Melia azadirachta bark (1) 50g of dried Melia azadirachta bark was mixed with benzene (500ml x 3) and methanol (500ml x
3) at room temperature for 24 hours, and the resulting extraction residue was extracted three times with 200 ml of hot water. The obtained extracts were combined and concentrated to dryness using a rotary evaporator to obtain 1960.5 mg of powder. (2) Dissolve 1000 mg of the powder obtained in (1) above in 200 ml of water, gradually add pure ethanol to the resulting aqueous solution at room temperature while stirring, and when the ethanol concentration in the aqueous solution reaches 80%. The addition was stopped, and the formed precipitate was collected by centrifugation to obtain 594.5 mg of brown powder. (3) 500 mg of the powder obtained in (1) above was dissolved in 50 ml of water, and this aqueous solution was poured into Spectra Pore 6 (molecular weight cut off: 50,000) and dialyzed against water. The dialyzed fluid was concentrated to dryness using a rotary evaporator to obtain 310 mg of brown powder. Example 1020 mg of Melia azadirachta bark hot water extract obtained in Reference Example (2) or (3) above was dissolved in 20 ml of distilled water, and a column (diameter 7.0 cm, length 35.0 cm) packed with Cephadex G-100 was prepared. cm) and gel filtration was performed using distilled water. When gel filtration is performed while quantifying the sugar content in the eluate using the phenol-sulfuric acid method, the polysaccharide is divided into three fractions. 184 mg of polysaccharide is obtained from the third eluting fraction. Next, dissolve 50 mg of this in 5 ml of distilled water, and add it to a column (diameter 4.0 cm, length
50.0cm) and gel filtration was performed using distilled water. The eluate was freeze-dried to obtain 2 mg of the desired anti-inflammatory polysaccharide. This was confirmed to be a single compound by high performance liquid chromatography and electrophoresis. As a result of pharmacological tests, it was confirmed that the polysaccharide of the present invention has a remarkable inhibitory effect on carrageenan edema. Next, a test example will be shown. Test Example 1 Inhibitory effect on carrageenan edema The inhibitory effect on carrageenan edema was measured using the polysaccharide of the present invention obtained in the example and prednisolone and indomethacin as controls. The sample was orally administered to ddY mice, and 0.05 ml of 1% carrageenan was injected into the right hind footpad one hour after administration, and the edema volume of the same footpad was measured over time. The edema suppression rate was determined from the ratio to the edema volume when no sample was administered. The results are shown in Table 1. From Table 1, it is clear that the polysaccharide of the present invention has an effect of suppressing inflammation caused by carrageenan.

[Table] Test Example 2 Acute Toxicity An acute toxicity test was conducted by administering the polysaccharide of the present invention to ICR male mice weighing 20±1 g.
The LD ₅₀ value was 460 mg/Kg or more for intraperitoneal administration and 1000 mg/Kg or more for oral administration. As is clear from the results of the pharmacological tests mentioned above,
The polysaccharide of the present invention has remarkable anti-inflammatory properties and extremely low toxicity, so it has excellent properties as an anti-inflammatory agent. The polysaccharide of the present invention is effective against various inflammatory diseases, and the dosage varies depending on symptoms, age, body weight, etc., but is usually 1 to 50 mg per day for adults.
It can be administered in 1 to 4 doses. The polysaccharides of the present invention are formulated for oral or parenteral administration using any required pharmaceutical carriers or excipients. Tablets, powders, capsules, granules, etc. for oral administration contain conventional excipients such as calcium carbonate, calcium phosphate, corn starch, potato starch, sugar, lactose, talc, magnesium stearate, gum arabic, etc. Good too. Liquid preparations for oral administration may be aqueous or oily suspensions, solutions, syrups, elixirs, and the like. Injectable preparations are in the form of solutions or suspensions;
It may contain formulation agents such as suspending, stabilizing or dispersing agents, sterile distilled water, essential oils such as peanut oil, corn oil or non-aqueous solvents;
It may contain polyethylene glycol, polypropylene glycol, etc. For rectal administration, it is provided in the form of a suppository composition, which may contain peripheral pharmaceutical carriers such as polyethylene glycol, lanolin, coconut oil, and the like. Furthermore, it can also be administered in the form of conventional external preparations such as ointments, external creams, lotions, and pasta. Specific Effects of the Invention As detailed above, according to the present invention, firstly,
Anti-inflammatory polysaccharides are provided. This polysaccharide is a new compound described in the literature, and as shown in the test examples, it exhibits a strong inhibitory effect on carrageenan edema and has very low toxicity, making it useful as an anti-inflammatory agent. According to the present invention, secondly, a method for producing the above polysaccharide is provided. That is, the polysaccharide is obtained by treating a hot water extract of Melia azadirachta bark with a molecular sieve and collecting a compound having a molecular weight of about 8,000. According to the present invention, thirdly, an advantageous method for producing the above polysaccharide is provided. That is, the polysaccharide undergoes the above molecular sieve treatment to have a molecular weight cut-off of approximately 1×10 ³ to 1×10 ⁵ to 1
High purity can be obtained by using a molecular sieve of ×10 ³ to 2 × 10 ⁵ and purifying the resulting fraction with an alkylated dextran gel. According to the present invention, fourthly, a more advantageous method for producing the above polysaccharide is provided. That is, the polysaccharide is
It can be obtained more easily by performing gel filtration using a gel filtration agent as the molecular sieve treatment. According to the present invention, fifthly, a more advantageous method for producing the above polysaccharide is provided. That is, the polysaccharide is
High purity can be easily obtained by using dextran gel, polyacrylamide gel, hydrophilic polyvinyl gel or porous glass beads as a gelling agent.

Claims (1)

[Claims] 1. Melia azadirachta (Melia azadirachta)
azadirachta) is an anti-inflammatory polysaccharide obtained from the bark of the tree with the following properties. (a) Color and shape White or slightly brown powder (b) Solubility Soluble in water, insoluble in organic solvents such as methanol, ethyl acetate, and benzene. (c) Sugar composition Consists of glucose, arabinose and fucose, with a composition ratio of 6:1:1. (d) Molecular weight approximately 8000 (e) Specific rotation [α] ²² _D : 16.7° (c = 0.5, H ₂ O) (f) Ultraviolet absorption spectrum When measured in an aqueous solution, it does not show an absorption maximum, but shows terminal absorption. . 2. A method for producing an anti-inflammatory polysaccharide having the following properties, which comprises treating a hot water extract of Melia azadirachta bark with a molecular sieve to collect a compound having a molecular weight of about 8,000. (a) Color and shape White or slightly brown powder (b) Solubility Soluble in water, insoluble in organic solvents such as methanol, ethyl acetate, and benzene. (c) Sugar composition Consists of glucose, arabinose and fucose, with a composition ratio of 6:1:1. (d) Molecular weight approximately 8000 (e) Specific rotation [α] ²² _D : 16.7° (c = 0.5, H ₂ O) (f) Ultraviolet absorption spectrum When measured in an aqueous solution, it does not show an absorption maximum, but shows terminal absorption. . 3 In the above molecular sieve treatment, first, the molecular weight cut-off is 1×10 ³
~1×10 ⁵ to 1×10 ³ to 2×10 ⁵ molecular sieves were used, and of the three fractions of polysaccharide obtained, the third eluting fraction was collected and further eluted. 3. The method for producing an anti-inflammatory polysaccharide according to claim 2, which is purified using an alkylated dextran gel. 4. The method for producing an anti-inflammatory polysaccharide according to claim 2 or 3, wherein the molecular sieve treatment is gel filtration, and the molecular sieve agent is a gel filtration agent. 5. Claim 4, wherein the gel filtration agent is dextran gel, polyacrylamide gel, hydrophilic polyvinyl gel, or porous glass beads.
A method for producing an anti-inflammatory polysaccharide as described in Section 1.