JPS6153337B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION Technical Field 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. 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. That is, 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 1×10 ³ to 1 ×10 ⁵ to 1 × 10 ³ to 2 × 10 ⁵ gel filtration agents [e.g., Cephadex G100 (trade name, Pharmacia product), Biogel P-100
(trade name, product of BioRad), etc.], and the first fraction of the three polysaccharide fractions is collected. Although this method is excellent in obtaining highly pure polysaccharide N9GI, it is extremely difficult to implement on a large scale due to complicated operations.
Furthermore, the gel filtration agent described above lacks mechanical strength and is therefore 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 that can be carried out on a large scale with simple operations. The object of the invention is achieved by the method described below. (1) Extract Melia azadirachta bark with hot water,
The extract is purified by a dialysis membrane method, an alcohol precipitation method, or an ultrafiltration method, and the resulting purified extract has a molecular weight fraction with an upper limit of 700 to 5000 and a lower limit of 100.
A method for producing polysaccharide N9GI having the following physicochemical properties, which comprises bringing the polysaccharide into contact with the following cross-linked dextran gel and collecting the polysaccharide from the contact aqueous solution. (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). IR ν ^KBr _nax cm ^-1 :3400, 1630, 1030 (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. (2) The molecular weight cutoff range of the dextran gel is 0.
700. The method for producing polysaccharide N9GI according to item 1, which has a molecular weight of 700. (3) The molecular weight cutoff range of the dextran gel is 0.
The method for producing polysaccharide N9GI according to item 1, wherein the polysaccharide N9GI has a molecular weight of 1,500 to 1,500. (4) The molecular weight cut-off range of the dextran gel is
100-5000, the method for producing polysaccharide N9GI according to item 1. DETAILED DESCRIPTION OF THE INVENTION According to the present invention, polysaccharide N9GI is obtained by extracting Melia azadirachta bark with hot water and purifying the extract by a dialysis membrane method, an alcohol precipitation method, or an ultrafiltration method. It is obtained by contacting the purified extract with a cross-linked dextran gel having a molecular weight cut-off of 700 to 5000 at the upper limit and 100 or less at the lower limit, and collecting the polysaccharide from the aqueous solution after the contact, that is, the contact aqueous solution. 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 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 done in a boiling water bath or over an open flame. 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 complete,
A hot water extract is obtained by filtering the extraction mixture. 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, and nonpolar organic solvents such as benzene, toluene, xylene, n-hexane, chloroform, carbon tetrachloride, and ethyl acetate. can be given. 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 a 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 Clearance TE-15 (product name), Amicon's
HIP5 (trade name, molecular weight cutoff 5000) or HIP10
(trade name, molecular weight cut off 10,000) 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 manufactured by Toyo Soda Kogyo Co., Ltd.
-UF membrane: TS-10 (molecular weight cut off 10000), TS-30
(30,000), TS-50 (50,000) and Amicon product ultrafiltration membrane: 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. 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.
It is. 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, the purified extract is brought into contact with a cross-linked dextran gel having a molecular weight cut-off range of upper limit 700 to 5000 and lower limit 100 or less, impurities are adsorbed to the gel, and polysaccharides are collected from the contact aqueous solution. This yields the desired polysaccharide N9GI. The gel used as an adsorbent in the present invention is a crosslinked dextran gel that has excellent mechanical strength and can be used for large-scale purification. Dextran gels that are highly crosslinked are particularly preferred in terms of mechanical strength. Although the molecular weight cut-off range of the gel is not critical, it is appropriate that the upper limit is 700 to 5000 and the lower limit is 100 or less. Examples of such gels include Sephadex G-10 (molecular weight cut-off range of 700 or less), G-
15 (1500 or less) and G-25 (100-5000). This step is carried out by passing the purified extract through a column filled with the gel, eluting with water, and evaporating or freeze-drying the eluate.
In this treatment, impurities are adsorbed to the gel, and the desired polysaccharide is not adsorbed. Therefore, this step can also be carried out in batches. That is, the desired polysaccharide can be obtained by stirring the purified extract together with the gel, then removing the gel by filtration or centrifugation, and evaporating or freeze-drying the filtrate. 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). IR ν ^KBr _nax cm ^-1 :3400, 1630, 1030 (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. 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
It is commercially available under product names such as Toyo Pearl HW-60 (product name, Toyo Soda Kogyo Co., Ltd., Japan) and Toyo Pearl HW-60 (product name, Toyo Soda Kogyo Co., Ltd., Japan). 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 and insoluble in organic solvents such as methanol, ethanol, acetone, ether, chloroform, ethyl acetate, 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) Infrared absorption spectrum As shown in Figure 2. IR ν ^KBr _nax cm ^-1 : 3400, 2930, 1620 (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 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 in which the composition ratio of glucose, arabinose and fucose is 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. IR ν ^KBr _nax cm ^-1 :3400, 2930, 1630 (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 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 N9GIb and N9GIb, and it is practical to use a mixture of the two, that is, in the present invention, the 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 determining 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
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 1 200 gr of Melia azadirachta bark was extracted three times with 2 parts of methanol each, and then extracted by boiling for 3 hours with 2 parts of distilled water three times. Collect the hot water extract and use approx.
Concentrate under reduced pressure to 500ml. It is then dialyzed against distilled water using a Spectrapore 2 dialysis membrane.
This internal solution is centrifuged to remove insoluble materials and concentrated to approximately 450 ml. Add twice the amount of ethanol to this, stir well, and then leave in a cold room (4°C) overnight.
The precipitate is collected by centrifugation, washed with 67% and then 100% ethanol, and dehydrated and dried with acetone and ether to obtain 3.47 gr of light brown powder. This 76.5mg
Dissolve in 5 ml of distilled water and add Sephadex G-25.
When applied to a column (φ2.5 x 45cm) packed with 2.5 cm and eluted with distilled water, the impurities are adsorbed to the column and the polysaccharide N9GI is eluted as it is, so the eluate is collected, concentrated, and freeze-dried, resulting in a polysaccharide N9GI of 28.9 mg
can get. Example 2 In Example 1, when the hollow fiber dialyzer Crillance TE15 (Terumo) was used instead of Spectrapore 2, the ethanol precipitate was
3.06gr was obtained, and this was also
When polysaccharide N9GI is purified at -25, 32.6 mg is obtained from 85.3 mg.
mg of polysaccharide N9GI is obtained. Example 3 Instead of using the Cephadex G-25 column in Example 1, 250 mg of the ethanol precipitate was dissolved in 100 ml of distilled water, and the Cephadex G-25 column was dissolved in 100 ml of distilled water.
Add 50ml, stir, and leave for 30 minutes to 1 hour. Filter or centrifuge to remove Cephadex G-25,
When the supernatant was concentrated and lyophilized, the polysaccharide N9GI was
90.3mg is obtained. When the polysaccharide N9GI of Examples 1, 2, and 3 is subjected to gel filtration using Sephadex G-100, it is confirmed that it contains only the polysaccharide N9GI fraction. That is, 160 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. 69 mg of polysaccharide N9GIa is obtained from the first eluted fraction, and 61 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 upper limit of the molecular weight cut-off range of the hot water extract of Melia azadirachta bark is
The desired polysaccharide N9GI can be obtained with high purity simply by contacting it with a crosslinked dextran gel having a molecular weight of 700 to 5000 and a lower limit of 100 or less and collecting the polysaccharide from the contact aqueous solution. 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,
The extract is purified by a dialysis membrane method, an alcohol precipitation method, or an ultrafiltration method, and the purified extract obtained is converted into a cross-linked dextran gel having a molecular weight cut-off range with an upper limit of 700 to 5000 and a lower limit of 100 or less. A method for producing polysaccharide N9GI having the following physicochemical properties, which comprises contacting with a polysaccharide and collecting the polysaccharide from a contact aqueous solution. (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). IR ν ^KBr _nax cm ^-1 :3400, 1630, 1030 (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. 2 The molecular weight cutoff range of the dextran gel is 0
-700 polysaccharide according to claim 1
Manufacturing method of N9GI. 3 The molecular weight cutoff range of the dextran gel is 0.
-1500 polysaccharide according to claim 1
Manufacturing method of N9GI. 4 The molecular weight cut-off range of the dextran gel is
100 to 5000, the method for producing polysaccharide N9GI according to claim 1.