JPS6153326B2 - - 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, fruits, roots, 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. The present inventors have previously discovered that a hot water extract of Melia azadirachta bark has an antitumor effect, and that a polysaccharide obtained by purifying the above extract by a specific method
We found that N9GI has even stronger antitumor effects. According to the above method, polysaccharide N9GI is obtained by purifying a hot water extract of Melia azadirachta bark by alcohol precipitation method or dialysis membrane method, dissolving the obtained purified product in water, and dissolving the aqueous solution with a molecular weight cut-off of approximately 1×
Using a gel filtration agent of 10 ³ to 1 × 10 ⁵ to 1 × 10 ³ to 2 × 10 ⁵ [e.g., Cephadex G100 (trade name, product of Pharmacia), Biogel P-100 (trade name, product of BioRad), etc.] It is obtained by gel filtration, and of the three polysaccharide fractions, the first fraction is collected. This method produces highly pure polysaccharide N9GI.
However, the complicated operations make it extremely difficult to implement on a large scale, and the gel filtration agent lacks mechanical strength, making it unsuitable 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. (1) Extract Melia azadirachta bark with hot water,
A method for producing polysaccharide N9GI having the following physicochemical properties, which comprises ultrafiltering the extract, adding a lower alcohol to the obtained ultrafiltration liquid, and collecting the resulting precipitate. (b) Color and shape The lyophilized product is a white powder or a pale yellowish brown powder. (b) Infrared absorption spectrum As shown in Figure 1 (polysaccharide obtained in Example 1). IRν ^KBr _nax cm ^-1 : 3400, 1630, 1030 (c) Ultraviolet absorption spectrum When measured in an aqueous solution, it shows no absorption maximum but only terminal absorption. (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 blue color when iodine is added. (2) The ultrafiltration has a molecular weight cutoff of about 10,000 to about
The method for producing polysaccharide N9GI according to item 1, which is ultrafiltration using a 50,000 ultrafiltration membrane. (3) The first alcohol is ethanol.
A method for producing polysaccharide N9GI according to Section 1 or 2. DETAILED DESCRIPTION OF THE INVENTION According to the present invention, polysaccharide N9GI is obtained by extracting the bark of Melia azadirachta with hot water, ultrafiltering the extract, and adding a lower alcohol to the obtained ultrafiltration liquid. It is produced by collecting the resulting precipitate. 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.
It is a woody plant that grows over 10 meters in height and grows naturally in tropical regions. In the method of the present invention, a hot water extract of 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 conveniently determined according to the quality of the raw materials and is usually 1 to 8 hours. After the extraction is completed, an extract is obtained by filtering the extraction mixture. Prior to the above hot water extraction treatment, 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 methanol, ethanol, propanol,
Examples include polar organic solvents such as pyridine and acetone, and non-polar organic solvents such as benzene, toluene, xylene, n-hexane, chloroform, carbon tetrachloride, and ethyl acetate. The hot water extract thus obtained is subjected to ultrafiltration as it is without being concentrated. Ultrafiltration uses an ultrafiltration membrane with a molecular weight cut-off of approximately 10,000 to 50,000.
It is carried out under pressure according to conventional methods. 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 the one manufactured by Toyo Soda Kogyo Co., Ltd.
TSK-UF membrane: TS-10 (molecular weight cut off 10000), TS
-30 (30,000), TS-50 (50,000) and Amicon product ultrafiltration membranes: YM10 (molecular weight cut off)
10000), PM10 (10000), YM30 (30000),
PM30 (30,000 yen) and XM50 (50,000 yen) are included. Especially TS-50 (Toyo Soda Kogyo product)
is preferred. Pressure during filtration is approximately 0.1 to 2 kg/
^cm2 is appropriate. The above ultrafiltration simultaneously performs concentration of the hot water extract and removal of low molecular weight impurities. The concentration of the concentrate is 5 to 20 mg/ml, preferably 10 to 20 mg/ml.
It is 15mg/ml. Next, the filtration liquid (concentrated liquid) obtained in this way
The desired polysaccharide is obtained by adding a lower alcohol and collecting the resulting precipitate according to a conventional method.
N9GI is obtained. Examples of lower alcohols used to form the precipitate include, in particular, lower alkyl alcohols such as methanol, ethanol, n-propanol, n-
Examples include butanol, but ethanol is most desirable. Precipitate formation is suitably carried out by adding alcohol in an amount twice the amount of the internal solution and leaving the mixture at a low temperature (0 to 6° C.) for several hours to overnight. The produced precipitate is collected by a conventional method, for example, by centrifugation, freeze-drying, etc. The precipitate thus collected is purified by dissolving it in water and reprecipitating it with a lower alcohol, if necessary. The polysaccharide N9GI thus obtained has the following physicochemical properties. (b) Color and shape The lyophilized product is a white powder or a pale yellowish brown powder. (b) Infrared absorption spectrum As shown in Figure 1. (Polysaccharide obtained in Example 1). IRν ^KBr _nax cm ^-1 : 3400, 1630, 1030 (c) Ultraviolet absorption spectrum When measured in an aqueous solution, it shows no absorption maximum but only terminal absorption. (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, if the polysaccharide obtained above is applied to a column packed with a gel filtration agent with a molecular weight cut-off of about 1 x 10 ³ - 2 x 10 ⁵ to 1 x 10 ³ - 8 x 10 ⁵ and eluted with distilled water, The polysaccharide is fractionated into two fractions. Let the first eluted fraction be polysaccharide N9GIa, and the later eluted polysaccharide N9GIb. As the gel filtration agent, dextran gel, polyacrylamide gel, polyvinyl polymer gel, porous glass beads, etc. are used. These are, for example, Sephadex G-
200, Cefacryl S-300 (product name, Pharmacia product, Sweden), Biogel P-300 (product name, Biorad product, USA), Toyopearl
HW-60 (Product name, Toyo Soda Kogyo Co., Ltd. product, Japan)
It is commercially available under product names such as. The structures and physicochemical properties of polysaccharide N9GIa and polysaccharide N9GIb are as follows. Polysaccharide N9GIa (a) Structure A neutral polysaccharide in which arabinose is α-(1→6) linked to the main chain of α-(1→4)-glucan, and the composition ratio of glucose and arabinose is 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 34000. (F) Infrared absorption spectrum As shown in Figure 2. IRν ^KBr _nax cm ^-1 : 3400, 2930, 1620 (g) Ultraviolet absorption spectrum When measured in an aqueous solution, it shows no absorption maximum, but only terminal absorption. (H) ¹³ C nuclear magnetic resonance spectrum The 100 MHz ¹³ C nuclear magnetic resonance spectrum measured in heavy water using TMS (tetramethylsilane) as an external standard is shown in Figure 3. Polysaccharide N9GIb (a) Structure The main chain is α-(1→4)-glucan, contains β-(1→3) fucose in the main chain, and has α-(1→6) arabinose as a branch. A neutral polysaccharide with a composition ratio of glucose, arabinose and fucose of approximately 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 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 21000. (F) Infrared absorption spectrum As shown in Figure 4. IRν ^KBr _nax cm ^-1 : 3400, 2930, 1630 (g) Ultraviolet absorption spectrum When measured in an aqueous solution, it does not show an absorption maximum, but shows only terminal absorption. (H) ¹³ C nuclear magnetic resonance spectrum The 100 MHz ¹³ C nuclear magnetic resonance spectrum measured in heavy water using TMS (tetramethylsilane) as an external standard is shown in Figure 5. 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 has a remarkable inhibitory effect on Sarcoma 180 solid mouse transplant tumor and female A solid mouse transplant tumor. It was confirmed that the 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 N9GIa and polysaccharide N9GIa.
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 9.5m
The sample was dissolved in PBS containing M 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. 4 0.1 ml of this cell suspension
Cells were subcutaneously transplanted into the back of ~5-week-old male ICR mice using a syringe. Therefore, the number of transplanted cells per animal was 1.0×10 ⁷ cells. (Sample administration) 1 from day 6 after transplantation of Sarcoma 180 cancer cells
The sample prepared above was intraperitoneally administered to mice using a syringe once a day for 10 consecutive days. Ten mice were used per sample per concentration.
As a control, the above-mentioned PBS, 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. (Method for determining effectiveness) Cancer tissue that had grown on the 35th day after cancer cell transplantation was removed, and its weight was measured (average value for 10 animals per group). The effect was determined by calculating the ratio (T/C) between this weight and that of the control. The control cancer tissue weight is
It was 10.83g. 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 removed together with ascites, diluted appropriately with physiological saline,
The number of cells was adjusted to 1.0×10 ⁶ cells/ml.
0.1 ml of this cell suspension was subcutaneously transplanted into the back of a 5-week-old male Balb/C mouse using a syringe. The number of transplanted cells per animal was 1.0×10 ⁵ cells. The sample was prepared in the same manner as in Test Example 1, and was applied once a day from the 11th day.
It was administered intraperitoneally to mice for 10 consecutive days. Judgment was made by cutting out tumor tissue on the 21st day after tumor cell transplantation and measuring its weight. The results are shown in Table 2.

[Table] As is clear from Tables 1 and 2, the polysaccharide N9GI of the present invention has a strong inhibitory effect on Sarcoma 180 solid tumor and Female A fibrosarcoma. Test Example 3 Acute Toxicity An acute toxicity test was conducted by administering the purified extract 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 of the present invention has remarkable antitumor properties and extremely low toxicity, so 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 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, 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 Add 2 methanol to 200g of Melia azadirachta bark and extract at room temperature for 5 hours to overnight. Filter the oil extract and add methanol (2). After repeating this operation three times in total, distilled water from Step 2 is added to the bark and extracted by boiling for 2 to 3 hours. Collect the extract by filtration or centrifugation. Repeat the same operation 3 times in total to obtain 3 extracts, approximately 5.5
Collect. After this, if necessary, further centrifugation is performed to remove insoluble matter. 2.3 g of this hot water extract (which contains about 7.5 gr of hot water extract) was added to the TSK-UF membrane TS-50.
(molecular weight cut off 50000, product name, Toyo Soda Kogyo Co., Ltd. product) ultrafiltration device SC-60 (product name,
Perform ultrafiltration using Toyo Soda Kogyo Co., Ltd. product). First, after concentrating to about 100 to 150 ml,
Add 150 to 200 ml of distilled water to this, perform further ultrafiltration, and concentrate again to about 100 ml. After repeating this operation 5 to 10 times and adding 2 times the volume (i.e. 200 ml) of ethanol to 100 ml of the internal solution (in which 1.98 gr of solid matter is dissolved) and stirring thoroughly, After standing at a low temperature (0 to 6°C) for several hours to overnight, the precipitate is collected by a conventional method such as centrifugation. Dissolve the precipitate again in 70 ml of distilled water, add twice the volume (140 ml) of ethanol, and repeat the same operation for 1~
Repeat twice. The obtained precipitate was reduced to 67%, 75%,
After sequentially washing with 100% ethanol and dehydrating with acetone and ether, 805 mg of polysaccharide N9GI is obtained as a pale yellow to white powder. When this was subjected to gel filtration using Cephadex G-100, it was found that it contained almost nothing other than polysaccharide N9GI. For reference, 150 mg of the above polysaccharide N9GI was dissolved in 20 ml of distilled water, poured into a column (diameter 4.0 cm, length 50.0 cm) packed with Sephadex G-200, and gel filtration was performed using distilled water. When gel filtration is performed while quantifying the sugar content in the elution using the phenol sulfuric acid method, the polysaccharide is fractionated into two. 72 mg of polysaccharide N9GIa is obtained from the first eluted fraction, and 64 mg of polysaccharide N9GIb is obtained from the second eluted fraction. These polysaccharides
N9GIa and N9GIb were confirmed to be single compounds by high performance chromatography and electrophoresis. Specific Effects of the Invention As detailed above, according to the present invention, polysaccharide
A method for industrially producing N9GI is provided. The polysaccharide N9GI is a substance that has not been described in any literature, and as shown in the test examples, it exhibits strong inhibitory activity against Sarcoma 180 solid cancer and Female A fibrosarcoma, and has very low toxicity, making it useful as an antitumor agent. be.
According to the present invention, a method for easily producing such polysaccharide N9GI is provided. That is, the desired polysaccharide N9GI can be obtained with high purity simply by ultrafiltering a hot water extract of Melia azadirachta bark, adding a lower alcohol to the filtrate, and collecting the resulting precipitate. . As described above, the production method of the present invention does not require complicated operations and can be easily implemented on a large scale, so it is excellent as an industrial production method.

[Brief explanation of the drawing]

FIG. 1 shows the infrared absorption spectrum of the polysaccharide N9GI of the present invention (the polysaccharide obtained in Example 1).
FIG. 2 shows the infrared absorption spectrum of polysaccharide N9GIa, and FIG. 3 shows the nuclear magnetic resonance spectrum of the same polysaccharide. FIG. 4 shows the infrared absorption spectrum of polysaccharide N9GIb, and FIG. 5 shows the nuclear magnetic resonance spectrum of the same polysaccharide.

Claims (1)

[Claims] 1 Melia azadirachta bark is extracted with hot water,
A method for producing polysaccharide N9GI having the following physicochemical properties, which comprises ultrafiltering the extract, adding a lower alcohol to the obtained ultrafiltration liquid, and collecting the resulting precipitate. (b) Color and shape The lyophilized product is a white powder or a pale yellowish brown powder. (b) Infrared absorption spectrum As shown in Figure 1. (Polysaccharide obtained in Example 1). IRν ^KBr _nax cm ^-1 : 3400, 1630, 1030 (c) Ultraviolet absorption spectrum When measured in an aqueous solution, it shows no absorption maximum but only terminal absorption. (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. 2 The ultrafiltration has a molecular weight cutoff of about 10,000 to about
The method for producing polysaccharide N9GI according to claim 1, which is ultrafiltration using a 50,000 ultrafiltration membrane. 3. The polysaccharide according to claim 1 or 2, wherein the lower alcohol is ethanol.
Manufacturing method of N9GI.