JPH0335294B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND TECHNICAL FIELD OF THE INVENTION The present invention relates to a method for producing polysaccharide N9GI. More specifically, the present invention relates to a method for producing polysaccharide N9GI, which comprises purifying a hot water extract of Melia azadirachta bark by a specific method. The polysaccharide obtained by the production method of the present invention is useful as an antitumor 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, fruit, branches, root bark, or resin of Melia azadirachta with water or a hydrophilic solvent, or by pulverizing it (Japanese Patent Publication No. 1973- 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. In addition, the present inventors first added alcohol to a hot water extract of the bark of Melia azadirachta and collected the resulting precipitate (Japanese Patent Application Laid-open No. 176914/1983), or treated the hot water extract with a dialysis membrane. reported that an extract with antitumor activity could be obtained by collecting the active ingredient from dialysis fluid (Japanese Patent Application Laid-Open No. 176915/1983). As a result of further research, the present inventors dissolved the purified extract in water and converted the aqueous solution into a gel with a molecular weight cut-off of approximately 1×10 ³ to 1×10 ⁵ to 1×10 ³ to 2×10 ⁵ . Filtration agents [e.g. Cephadex G100 (trade name, Pharmacia product), Biogel P-100 (trade name,
Gel filtration using BioRad product) etc.
We learned that a novel polysaccharide N9GI can be obtained by collecting the first fraction among the three polysaccharide fractions. This method produces highly pure polysaccharides.
Although it is excellent in obtaining N9GI, it is very difficult to implement on a large scale due to complicated operations, and the above gel filtration agent lacks mechanical strength, so it is not very suitable for industrial implementation. . Purpose of the Invention Therefore, the present invention provides a polysaccharide suitable for industrial implementation.
The purpose is to provide a manufacturing method for N9GI. That is, an object of the present invention is to provide a method for producing polysaccharide N9GI using an apparatus that is easy to operate and can be carried out on a large scale. The object of the invention is achieved by the method described below. Melia azadirachta bark is extracted with hot water, the extract is purified by alcohol precipitation method, dialysis membrane method or ultrafiltration method, and the obtained extracted purified product is treated with a basic ion exchanger. A method for producing polysaccharide N9GI having the following physicochemical properties. (b) Color and shape The freeze-dried product is a white or pale yellowish brown powder. (b) Infrared absorption spectrum IRν ^KBr _nax cm ^-1 : 3400, 2920, 1630, 1400, 1360,
1150, 1070, 1030, 920, 840 (c) Ultraviolet absorption spectrum When measured in an aqueous solution, it does not show an absorption maximum and only shows absorption below. (d) Solubility Soluble in water and insoluble in organic solvents such as methanol, ethanol, acetone, ether, chloroform, ethyl acetate, benzene and hexane. (e) Color reaction It is positive for the phenol sulfuric acid reaction and the anthrone sulfuric acid reaction, and exhibits a bluish-green color when iodine is added. Detailed Description of the Invention The scientific name of Melia azadirachta, which is the raw material plant for the method of the present invention, is Melia azadirachta Linn. or Azadirachta indica.
Juss) is a woody plant that grows in tropical regions and can reach a height of over 10 meters. In the method of the invention, the bark of Melia azadirachta is used as a raw material. The extraction treatment of the bark with hot water is carried out according to a conventional method. That is, it is carried out by adding hot water to shredded bark, or by adding water to 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 extraction mixture is filtered to obtain a hot water extract. Prior to such hot water extraction, it is also desirable to pre-extract the bark with an organic solvent and/or water at room temperature to remove unnecessary components. Solvents used for extraction pretreatment include polar organic solvents such as methanol, ethanol, propanol, pyridine, and acetone, benzene,
Examples include nonpolar organic solvents such as toluene, xylene, n-hexane, chloroform, carbon tetrachloride, and ethyl acetate. Since the hot water extract thus obtained still contains a large amount of impurities, the extract is purified by alcohol precipitation, dialysis membrane method, or ultrafiltration method. When purifying by alcohol precipitation, an alcohol such as methanol, ethanol, or propanol is added to the above extract, and the resulting precipitate is collected by a conventional method, for example, by centrifugation. When purifying by the dialysis membrane method, the extract is placed in a dialysis membrane, dialyzed by immersion in water, and the dialyzed solution is concentrated to dryness or freeze-dried as desired to obtain an extract.
Dialysis membranes with a molecular weight cutoff of 50,000 or less, such as Spectra Pore 1-6 (trade name, manufactured by Spectrum Medical Industries),
Visking tube (trade name, Union Carbide product) is used. Alternatively, a hollow fiber dialyzer with a molecular weight cut-off of about 5,000 to 10,000 may be used. For example, Terumo Corporation's Clarins TE-15 (product name), Amicon's HIP5
(trade name, molecular weight cutoff 5000) or HIP10 (trade name, molecular weight cutoff 10000) can be used.
In order to increase the degree of purification, the above-mentioned dialysis membrane method and alcohol precipitation method may be combined. That is, a highly purified polysaccharide can be obtained by adding alcohol to the dialysis solution and collecting the resulting precipitate. When purifying by ultrafiltration method, the molecular weight cut-off is approx.
It is carried out under pressure according to a conventional method using an ultrafiltration membrane of 10,000 to 50,000. The material of the filtration membrane is not particularly limited as long as it has the above-mentioned molecular weight cutoff, but a synthetic polymer casted on a nonwoven fabric or the like is preferably used. An example of such a filtration membrane is TSK-UF manufactured by Toyo Soda Kogyo Co., Ltd.
Membrane: TS-10 (molecular weight cut off 10000), TS-30 (molecular weight cut off 10000)
30000), TS-50 (50000) and Amicon product ultrafiltration membrane: YM10 (molecular weight cut off 10000),
PM10 (10,000), YM30 (30,000), PM30 (30,000)
30,000) and XM50 (50,000).
Particularly preferred is TS-50 (manufactured by Toyo Soda Kogyo Co., Ltd.).
Appropriate pressure during filtration is approximately 0.1 to 2 kg/cm ² . Ultrafiltration simultaneously achieves concentration of the hot water extract and removal of low molecular contaminants. The concentration of the concentrate is 5 to 20 mg/ml, preferably 10 to 15 mg/ml. The precipitate obtained by the alcohol precipitation method or the solid obtained by drying the dialysis fluid in the dialysis membrane method is dissolved in water to obtain the purified extract of the present invention. The internal solution obtained by the dialysis membrane method or the ultrafiltration method is directly used as the purified extract of the present invention. Next, this purified extract is brought into contact with a basic ion exchanger, and impurities are adsorbed onto the exchanger. As the basic ion exchanger, for example, a strong basic ion exchanger having a quaternary amine as an exchange group such as QAE Sephadex A-50 (trade name, manufactured by Pharmacia) is used. The contact between the extract aqueous solution and the basic ion exchanger is
It is carried out in a batch manner or in a continuous manner using a column. The desired polysaccharide N9GI is collected from the contact aqueous solution by a conventional method, for example, by freeze-drying. Polysaccharide N9GI obtained by the method of the present invention has the following physicochemical properties. (b) Color and shape The freeze-dried product is a white powder or a pale yellowish brown powder. (b) Infrared absorption spectrum IRν ^KBr _nax cm ^-1 : 3400, 2920, 1630, 1400, 1360,
1150, 1070, 1030, 920, 840 (c) Ultraviolet absorption spectrum When measured in an aqueous solution, it does not show an absorption maximum and only shows absorption below. (d) Solubility Soluble in water and insoluble in organic solvents such as methanol, ethanol, acetone, ether, chloroform, ethyl acetate, benzene and hexane. (e) Color reaction It is positive for the phenol sulfuric acid reaction and the anthrone sulfuric acid reaction, and exhibits a bluish-green color when iodine is added. For reference, the polysaccharide N9GI obtained above has a molecular weight cut-off of approximately 1×10 ³ to 2×10 ⁵ to 1×10 ³ to 8×10 ⁵
The polysaccharide is separated into two fractions by applying it to a column packed with a gel filtration agent and eluting with distilled water. Let the first eluted fraction be polysaccharide N9GIa, and the later eluted polysaccharide N9GIb. The above gel filtration agents include dextran gel, polyacrylamide gel,
Polyvinyl polymer gel, porous glass beads, etc. are used. These include, for example, Cephadex G-200, Cephacryl S-300 (trade name, Pharmacia product, Sweden), Biogel P-
300 (product name, Biorad Co., Ltd. product, USA), Toyopearl HW-60 (product name, Toyo Soda Kogyo Co., Ltd. product,
It is commercially available under product names such as Japan). The structures and physicochemical properties of polysaccharide N9GIa and polysaccharide N9GIb are as follows. Polysaccharide N9GIa a) Structure A neutral polysaccharide with α-(1→6) arabinose linked to the main chain of α-(1→4)-glucan, with a composition ratio of glucose and arabinose of approximately 5:1. b) Color and shape The freeze-dried product is a white powder. c) Solubility Soluble in water, methanol, ethanol, acetone, ether, chloroform, ethyl acetate,
Insoluble in organic solvents such as benzene and hexane. d) Color reaction It is positive for the phenol sulfuric acid reaction and the anthrone sulfuric acid reaction, and exhibits a bluish-green color when iodine is added. e) Molecular weight It gives a single peak in Sephadex G-200 column gel chromatography, and the molecular weight is approx.
It is 94000. F) Specific rotation [α] ²⁵ _D : +136.0° (C=0.5, H ₂ O) G) Infrared absorption spectrum IRν ^KBr _nax cm ^-1 : 3400, 2930, 1620, 1410, 1370,
1260, 1150, 1080 h) Ultraviolet absorption spectrum When measured in an aqueous solution, it does not show an absorption maximum, but only shows terminal absorption. li) ¹³ C nuclear magnetic resonance spectrum The 100MHz ¹³ C nuclear magnetic resonance spectrum measured in heavy water using TMS (tetramethylsilane) as an external standard is as follows. δppm: 62.1, 62.7, 67.3, 72.9, 74.8, 78.1,
78.7, 82.4, 85.5, 99.2, 101.1, 108.9 Polysaccharide N9GIb a) Structure α-(1→4)-glucan as the main chain, containing β-(1→3) fucose in the main chain, as a branch A neutral polysaccharide containing α-(1→6) arabinose and having a composition ratio of glucose, arabinose and fucose of about 5:2:1. b) Color and shape The freeze-dried product is a white powder. c) Solubility Soluble in water, methanol, ethanol, acetone, ether, chloroform, ethyl acetate,
Insoluble in organic solvents such as benzene and hexane. d) Color reaction: Positive for phenol sulfuric acid reaction and anthrone sulfuric acid reaction, and exhibits blue-green color when iodine is added. e) Molecular weight It gives a single peak in Sephadex G-200 column gel chromatography, and the molecular weight is approx.
It is 21000. F) Specific rotation [α] ²⁵ _D : +143.7° (C=0.5, H ₂ O) G) Infrared absorption spectrum IRν ^KBr _nax cm ^-1 : 3400, 2930, 1630, 1420, 1260,
1080, 020, 810 h) Ultraviolet absorption spectrum When measured in an aqueous solution, it does not show an absorption maximum, but only shows terminal absorption. 1) ¹³ C nuclear magnetic resonance spectrum The MHz ¹³ C nuclear magnetic resonance spectrum measured in heavy water using TMS (tetramethylsilane) as an external standard is as follows. δppm: 18.2, 62.3, 62.7, 67.4, 71.1, 72.0,
73.2, 74.9, 78.3, 78.9, 82.9, 83.9, 85.6,
99.4, 101.2, 105.0, 109.1 The polysaccharide N9GI of the present invention can be considered to be a mixture of the above-mentioned polysaccharide N9GIa and polysaccharide N9GIb, and as a result of pharmacological tests, it was found that It was confirmed that it has a significant inhibitory effect. The above polysaccharides N9GIa and N9GIb also showed similar pharmacological activity. Therefore, when used as an antitumor agent, the polysaccharide N9GI of the present invention may be further combined with polysaccharide N9GI and polysaccharide N9GI.
It is not necessary to separate the polysaccharide from N9GIb, and it is practical to use a mixture of the two, that is, in the present invention, in the form of polysaccharide N9GI. Next, a test example of the polysaccharide N9GI of the present invention will be shown. Test example 1 Effect on Sarcoma 180 solid cancer (sample preparation) Phosphate buffered saline (manufactured by Gibco, phosphoric acid
Each fraction sample was dissolved in a solution of 0.5% carboxymethylcellulose (CMC) suspended in PBS containing 9.5mM to a predetermined concentration. (Sarcoma 180 cancer cell transplantation) Sarcoma subcultured in the peritoneal cavity of ICR mice
180 cancer cells were taken out together with ascites fluid and diluted appropriately with physiological saline so that the number of cells was 1.0 x 10 ⁸ cells/ml. 0.1ml of this suspension at 4 weeks of age
Cells were transplanted subcutaneously into the back of male ICR mice using a syringe. (Sample administration) Starting on the 6th day after transplantation of Sarcoma 180 cancer cells, 0.1 ml of the sample prepared above was administered into the abdominal cavity once a day for 10 consecutive days using a syringe. Six mice were used per sample per concentration. As a control, the above-mentioned PBS containing CMC, which was used as a solvent for the sample, was administered in the same manner. The dosage was expressed as mg per kg of mouse body weight. (Efficacy evaluation method) Cancer tissue that had grown on the 21st day after the transplantation of cancer cells was excised and its weight was measured (average value of 6 animals per group). The effect was determined by calculating the ratio (T/C) between this weight and that of the control. Control cancer tissue weights were 1.5-3.5 g. Ratio values of 100 to 71% are ineffective (-), 70 to 51% are slightly effective (+), 50 to 21% are effective (), and 20 to 0% are significantly effective ( ). The results are shown in Table 1.

[Table] Test Example 2 Effect on female A fibrosarcoma Female A subcultured in the peritoneal cavity of Balb/C mice
Fibrosarcoma (Meth A fibrosarcoma) cells were taken out together with ascites and diluted appropriately with physiological saline to adjust the number of cells to 1.0×10 ⁶ cells/ml. Add 0.1 ml of this cell suspension to 5-week-old male Balb/C.
It was transplanted subcutaneously into the back of a mouse using a syringe. The number of transplanted cells per animal was 1.0×10 ⁵ cells. The sample is
Prepared in the same manner as Test Example 1, and administered once a day from the 11th day.
It was administered intraperitoneally to mice twice for 10 consecutive days. Judgment was made by cutting out the tumor tissue on the 21st day after tumor cell transplantation and measuring its weight. The results are shown in Table 2.

[Table] (Note)
Comparative Example A and Comparative Example B are the same as in Table 1.
As is clear from Tables 1 and 2, the polysaccharide N9GI of the present invention has a strong inhibitory effect on Sarcoma 180180 solid tumor and Female A fibrosarcoma. Moreover, the efficacy was about 5 to 6 times stronger than that of Comparative Examples A and B, indicating that the purity of the active ingredient was higher. Test Example 3 Acute Toxicity An acute toxicity test was conducted by administering the polysaccharide N9GI of the present invention to ICR male mice weighing 20±1 g.
The LD ₅₀ value was 600 mg/Kg or more when administered intraperitoneally. As is clear from the results of the pharmacological tests mentioned above,
The polysaccharide N9GI of the present invention has remarkable antitumor properties,
Since it has extremely low toxicity, it has excellent properties as an anticancer agent. Since the polysaccharide of the present invention has a strong suppressive effect on established solid cancers, it is considered that the polysaccharide of the present invention has an immunostimulatory antitumor effect. The polysaccharide of the present invention is effective against various cancer diseases, and the dosage varies depending on symptoms, age, body weight, etc., but is usually 100 to 2,500 doses per day for adults.
mg, and can be administered in 1 to 4 divided doses. The polysaccharide N9GI of the present invention is 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, they may be provided in the form of suppository compositions and may contain well-known pharmaceutical carriers such as polyethylene glycol, lanolin, coconut oil, and the like. Next, the present invention will be explained in more detail with reference to Examples. Example 1 (1) 50 g of dried Melia azadirachta bark was mixed with benzene (500 ml x 3) and methanol (500 ml x 3).
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 coagulated to dryness using a rotary evaporator to obtain 310 mg of brown powder. (4) 200ml of 100mg of powder obtained in (2) or (3) above
dissolved in distilled water. Activated with 0.5M NaCl
QAE Cephadex A-50 was mixed with the above aqueous solution and stirred for 6 hours, followed by suction filtration. The filtrate was dialyzed and then lyophilized to obtain 20.5 mg of the desired polysaccharide N9GI. Example 2 100 mg of the powder obtained in Example 1 (2) or (3) was
Dissolved in 2000ml of distilled water. A column was filled with QAE Sephadex A-50 activated with 0.5 M NaCl, the above aqueous solution was passed through the column, and the effluent was collected, dialyzed, concentrated, and lyophilized to obtain 25.8 mg of the desired polysaccharide N9GI. Specific Effects of the Invention As detailed above, according to the present invention, polysaccharide
A method for industrially producing N9GI is provided. As shown in the test examples, polysaccharide N9GI exhibits strong inhibitory activity against Sarcoma 180 transplanted cancer and Female A fibrosarcoma, and has very low toxicity, making it useful as an antitumor agent. According to the present invention, a method for industrially advantageously producing such polysaccharide N9GI is provided.

Claims (1)

[Claims] 1. Melia azadirachta bark is extracted with hot water, the extract is purified by alcohol precipitation, dialysis membrane method, or ultrafiltration method, and the obtained extracted and purified product is purified with a basic ion exchanger. A method for producing polysaccharide N9GI having the following physicochemical properties, which comprises: (b) Color and shape The freeze-dried product is a white or pale yellowish brown powder. (b) Infrared absorption spectrum IRν ^KBr _nax cm ^-1 : 3400, 2920, 1630, 1400, 1360,
1150, 1070, 1030, 920, 840 (c) Ultraviolet absorption spectrum When measured in an aqueous solution, it shows only terminal absorption without an absorption maximum. (d) Solubility Soluble in water and insoluble in organic solvents such as methanol, ethanol, acetone, ether, chloroform, ethyl acetate, benzene and hexane. (e) Color reaction It is positive for the phenol sulfuric acid reaction and the anthrone sulfuric acid reaction, and exhibits a bluish-green color when iodine is added.