JPS6019718A - Preparation of polysaccharide n9gi - Google Patents

Abstract

PURPOSE:To obtain the titled polysaccharide useful as an antitumor agent simply in a large scale, by extracting bark of Melia azadirachata Linn. with hot water, purifying the extracted solution by dialysis membrane method, etc., bringing it into contact with specific dextran gel, collecting it from the contacted aqueous solution. CONSTITUTION:Bark of Melia azadirachata Linn is extracted with hot water, the extracted solution is purified by dialysis membrane method, alcohol precipitation method, or ultrafiltration. The prepared extracted solution is brought into contact with crosslinked dextran gel having 700-5,000 upper limit of fractionated molecular weight range and <=100 lower limit, and polysaccharide N9GI (lyophilized polysaccharide N9GI is white powder or light yellowish brown powder; soluble in water, insoluble in an organic solvent such as methanol, etc.; positive in phenol sulfuric acid reaction, etc. and addition of iodine shows blue green) is obtained from he contacted aqueous solution. The dextran gel used as an adsorbent is preferably required to have improved mechanical strength, and high crosslinking.

Description

[Detailed description of the invention] ■1 Background of the invention Skin Togane” 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.

It has been known that Melia Φ Azadirachta extract has various pharmacological actions. That is, a method for obtaining skin cosmetics by extracting the bark, leaves, flowers, eastern parts, branches, root bark or resin of Melia azadirachta with water or a hydrophilic solvent or by pulverizing them (Japanese Patent Publication No. 52-28853) .52-
28854 and 53-1Ii"ol 25),
Above method 1] Method for extracting A. azadirachta plant I with a wood-philic solvent and/or hot water to obtain components having antibacterial effects and gastrointestinal and liver function improving effects (Japanese Patent Publication No. 53-101
24) and a method for extracting the Melia azadirachta raw material with a hydrophobic solvent to obtain ingredients effective for the treatment of skin diseases and rheumatism (Japanese Patent Publication No. 53-13689) has been reported.

The present inventors have previously demonstrated that the hot water extract of Melia azadirachta bark has an antitumor effect, and that the polysaccharide N9GI obtained by purifying the oil extract by a specific method has an even stronger antitumor effect. I discovered that. That is, polysaccharide N9GI
The hot water extract of the bark of Melia azadirachta is purified by alcohol precipitation method or dialysis membrane method, the obtained purified product is dissolved in wood, and the aqueous solution is mixed with a molecular weight cut-off of about 5.times.10 to 1.times.10 to 3.5. Gel filtration is carried out using 1×10 to 2
It is obtained by collecting the first two fractions of the three polysaccharide fractions. 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, and the gel filtration agent lacks mechanical strength, making it difficult to implement in industrial applications. It is not very suitable for practical implementation.

II. OBJECTS OF THE INVENTION Therefore, the present invention provides polysaccharide N9GI suitable for industrial implementation.
The purpose is to provide a manufacturing method for.

That is, an objective 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) Melia azadirachta bark is extracted with hot water, the extract is purified by dialysis membrane method, alcohol precipitation method, or ultrafiltration method, and the obtained purified extract has a molecular weight cut-off range of 700 to 700. 5,000 with a lower limit of 100 or less, and collecting the polysaccharide from the contact aqueous solution.

(b) Color and shape The lyophilized product is a white powder or a tan powder.

(b) Infrared absorption spectrum as shown in Figure 1 (polysaccharide obtained in Example)
.

(c) Ultraviolet absorption spectrum measured in an aqueous solution 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) Fraction molecule # range of the dextran gel is 0 to 7
00. The method for producing polysaccharide N9GI according to item 1.

(3) The molecular weight cutoff range of the dextran gel is θ~1
, 500. The method for producing polysaccharide N9GI according to item 1.

(4) The molecular weight cutoff range of the dextran gel is 100
The method for producing polysaccharide N9GI according to item 1, wherein the polysaccharide N9GI has a molecular weight of 5,000.

■1 Detailed Description of the Invention According to the present invention, polysaccharide N9GI can be obtained by extracting Melia azadirachta bark with hot water and purifying the extract by dialysis membrane method, alcohol precipitation method or ultrafiltration method. The upper limit of the molecular weight cut-off range is 700 to 5,000.
The polysaccharide is obtained by contacting with a cross-linked dextran gel having a lower limit of 100 or less, and collecting the polysaccharide from the aqueous solution after contact, that is, the contact aqueous solution.

Melia azadirachta, which is the raw material plant for the method of the present invention, has the scientific name Melia azadirachta red linnaeus (-moon), and is a woody plant that grows naturally in tropical regions and reaches a height of 10 m or more. 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, 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, propatool, pyridine, and acetone, and nonpolar solvents such as benzene, toluene, xylene, n-hexane, chloroform, carbon tetrachloride, and ethyl acetate. Examples include organic solvents.

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 propatool 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 cut-off of 50,000 or less, such as Spectra Bore 1 to 6 (trade name, Spectrum
Medical Industries, Inc.), Visking
A tube (trade name, manufactured by one company, Union Carby) is used. Alternatively, the molecular weight cut-off is 5,000 to 10.0
A hollow fiber type dialyzer of about 0.00 may be used.

For example, Crillance TE-15 (trade name) manufactured by Chil-Nana Co., Ltd., HIP5 (trade name, molecular weight cutoff 5
,000) or HIPIO (trade name, molecular weight cut off 1
0,000) can be used. In order to reduce the degree of purification, it is also possible to combine the dialysis membrane method and the alcohol precipitation method described above. 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, an ultrafiltration membrane with a molecular weight of about 10,000 to 50,000 is used.
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 membrane made by casting a synthetic polymer onto a nonwoven fabric or the like is preferably used. An example of such a filtration membrane is the TSK-UF membrane: TS-10 manufactured by Toyo Soda Tgyo Co., Ltd.
(molecular weight cut off 10.000), TS-30 (molecular weight cut off 30.0
00), TS-50 (50,000) and Amicon product ultrafiltration s: :jFto (fractionated molecules it+o, ooo, +, PM to (10,
000), YM 30 (30,000),
PM30 (30,000) and XM50 (50,000)
000). Particularly preferred is TS-50 (manufactured by Toyo Soda Kogyo Co., Ltd.). Pressure during filtration is approximately 0.1~
2 kg/cm is suitable. Ultrafiltration simultaneously achieves concentration of the hot water extract and removal of low molecular contaminants. The concentration of the concentrate is 5-20 mg/m,
The preferred amount is 10 to 15 mg/m.

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 upper limit of the molecular weight cut-off range was 7.
The desired polysaccharide N9GI is obtained by contacting with a crosslinked dextran gel having a molecular weight of 00 to 5,000 and a lower limit of 100 or less to adsorb impurities to the gel, and collecting the polysaccharide from the contact aqueous solution.

The gel that can be used as an adsorbent in the present invention is a cross-linked dextran gel, which has low 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 suitable that the molecular weight range is from 700 to 5,000 and the lower limit is 100 or less. An example of such a gel is Sephadex G-10 (molecular weight cutoff range 700
(below), G-15 (1,500 or more), and G-25 (100 to 5,000). This step is carried out by passing the purified extract through a column packed with 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 the /Hetsch style. In other words, the purified extract
The desired polysaccharide can be obtained by stirring with the gel, then removing the gel by filtration or centrifugation, and evaporating the filtrate to dryness or freeze-drying.

1 The polysaccharide N9GI thus obtained has the following physicochemical properties.

(b) Color and shape The lyophilized product is a white powder or a tan powder.

(b) Infrared absorption spectrum as shown in Figure 1 (polysaccharide obtained in Example 1)
.

(c) Ultraviolet absorption spectrum measured in an aqueous solution 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 Positive 2 for phenol sulfuric acid reaction and anthrone sulfuric acid reaction The addition of iodine gives it a blue-green color.

For reference, the polysaccharide obtained above was fractionated with 3 5
3. The polysaccharide is fractionated into two fractions when applied to a column packed with about 1×10 to 2×10 to 1×10 to 8×10 gel filtration agent and eluted with distilled water. The polysaccharide that elutes first is N9GIa, and the polysaccharide that elutes later is N9GIb.
shall be. ” As the gel filtration agent, dextran gel,
Polyacrylamide gel, polyvinyl polymer gel, porous glass beads, etc. are used. These include Sephadex G-200, Sephacryl S-300 (
(Product name, Pharmacia product, Sweden), Biogel P-300 (Product name, Bio-Rad product, USA),
Toyo Pearl HW-60 (product name, Toyo Soda Kogyo 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.

' -N9GI a b) Structure: α-(1→4)-glucan has arabinose in its main chain.
A neutral polysaccharide with (1 → 6) bonds and a composition ratio of glucose and arabinose of about 5=1.

mouth) color and shape The lyophilized 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.

2) 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 has a molecular weight of about 94,000.

f) Specific rotation 2 [α) Knee 35° (c=0.4.HO)2 g) Infrared absorption spectrum As shown in FIG.

h) Ultraviolet absorption spectrum When measured in an aqueous solution, it does not show an absorption maximum, but only shows terminal absorption.

3 IJ) C Nuclear Magnetic Resonance Spectrum The loOMHz C nuclear magnetic resonance spectrum measured in heavy water using TMS (tetramethylsilane) as an external reference is shown in FIG.

' N 9 G Engineering b) Structure α-(1-4)-glucan is the main chain, β-(l→3) fucose is contained in the main chain, and α-(1-4)-glucan is the main chain.
→6) A neutral polysaccharide containing arabinose and having a composition ratio of glucose, arabinose and fucose of approximately 5:2:1.

mouth) color and shape The lyophilized 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.

2) 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 has a molecular weight of about 21,000.

f) Specific rotation 6 g) Infrared absorption spectrum As shown in FIG.

KBr-] I R11Cm: 3400, 2930.1830a
x H) Ultraviolet absorption spectrum When measured in an aqueous solution, it does not show an absorption maximum, but only shows terminal absorption.

3) C nuclear magnetic resonance spectrum The 100MH2C nuclear magnetic resonance spectrum measured in heavy water using TMS (tetramethylsilane) as an external reference is shown in Figure 5.

The polysaccharide N9GI of the present invention can be considered to be a mixture of the above-mentioned polysaccharide NQGIa and polysaccharide N9GI b, and as a result of pharmacological tests, it was found that the polysaccharide N9GI has a significant effect on Sarcoma 180 solid mouse transplanted tumor and female A solid mouse transplanted tumor. It was confirmed that it has an inhibitory effect. In addition, 2nd polysaccharide N9G
Ia 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 N9GI.
It is not necessary to separate into b and b, but in the state of a mixture of both,
That is, it is practical to use the polysaccharide N9G■ in the present invention.

Next, a test example of the polysaccharide N9GI of the present invention will be shown.

Test Example 1 Sarcoma 1801 Chiganni Rumu (Sample Preparation) A sample was dissolved in phosphate buffered saline (manufactured by Gibco, PBS containing 9.5 mM phosphoric acid) to a predetermined concentration.

(Transplantation of Sarcoma 180 Gaff Cells) Sarcoma 180 cancer cells subcultured in the peritoneal cavity of ICR mice were taken out together with ascites fluid, and diluted appropriately with physiological saline until the number of cells reached 1. , OXlO pieces/m. 4 to 0.1 mJ1 of this cell suspension
Cells were subcutaneously transplanted into the back of a 5-week-old male ICR mouse using a syringe. Therefore, the number of transplants per animal is 1.0Xl.
There are 7 pieces.

(Sample injection) The sample prepared above was injected into the abdominal cavity of a mouse using a syringe once a day for 10 consecutive days starting 6 days after implantation of Sarcoma 180 cancer cells.

Ten mice were used per sample liter 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 in mg per kg of mouse body weight.

(Method for determining effectiveness) Cancer tissue that grew 351 days after cancer cell transplantation was removed,
The weight was measured (average value of 10 animals per group). The effect was determined by taking the ratio (T/C) between this weight and that of the control. The cancer tissue weight of the control was 910.83 g. The results are shown in Table 1.

Table 1 Test Example 2 Female A11l subcultured in the peritoneal cavity of Ba1b/C mice that gain weight in the evening
Meth A fibrosarcoma cells were taken out together with ascites and diluted appropriately with physiological saline to adjust the number of cells to ioxto cells/m 5L. A 0.1 m cell suspension of this cell suspension was added to a 5-week-old male Ba.
It was transplanted subcutaneously to the back of 1b/C mice using a syringe. −
The number of transplanted cells per mouse is 1.0×10 cells.

The sample was prepared in the same manner as in Test Example 1, and the sample was prepared in the same manner as in Test Example 1.
It was administered intraperitoneally to mice once a day for 10 consecutive days. Judgment was made by cutting out a heavy tumor tissue on the 21st day after tumor cell implantation and measuring its weight. The results are shown in Table 2.

Table 2: Risk rate determined by t-test As is clear from Tables 1 and 2, the polysaccharide N9 of the present invention
GI has a strong inhibitory effect on Sarcoma 180 solid tumor and Female A fibrosarcoma.

Test Example 3 An acute toxicity test was conducted by administering the extracted and purified product of the present invention to ICR male mice weighing 20±Ig. As a result, the LD value was 0. .

As is clear from the results of the above pharmacological tests, 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,
The dosage varies depending on symptoms, age, body weight, etc., but is usually 100 to 2,500 mg per day for adults, 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.

Preparations for injection are in the form of solutions or suspensions and may contain formulating agents such as suspending, stabilizing or dispersing agents, sterile distilled water, essential oils such as bean oil, corn oil or non-aqueous oils. It may contain a solvent, polyethylene glycol, polypropylene glycol, etc.

For rectal administration, it may be provided in the form of a halved composition 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 3 times with 2 portions of methanol each, and then boiled for 3 times with 2 portions of distilled water for 3 hours. The hot water extracts are collected and concentrated under reduced pressure to approximately 500 mJl. It is then dialyzed against distilled water using a Spectrabore 2 dialysis membrane. This internal solution is centrifuged to remove insoluble matter and concentrated to about 450 mM. Add twice the amount of ethanol to this, stir well, and then store in a cold room (4℃).
I will set it up in the evening. The precipitate was collected by centrifugation, 67% then 10
After washing with 0% ethanol and dehydrating and drying with acetone and ether, 3.47 gr of light brown powder is obtained. This 7
Dissolve 6.5 mg in 5 mJl of distilled water, apply it to a column (φ2.5 x 45 cm) packed with Sephadex G-25, and elute with distilled water. Impurities will be adsorbed to the column and polysaccharide N9GI will be eluted as it is. The eluate is collected, concentrated, and freeze-dried to obtain 2'8.9 mg of polysaccharide N9GI.

Example 2 In Example 1, when the hollow fiber dialyzer Crillance TE15 (Terumo) was used instead of Spectrapore 2, 3.06 gr of ethanol precipitate was obtained, which was also treated with Sephadex G-25. When polysaccharide N9GI is purified, 32.6 mg of polysaccharide N is extracted from 85.3 mg.
9GI is obtained.

Example 3 Instead of using a Sephadex G-25 column in Example 1, 250 mg of ethanol precipitate was dissolved in 100 ml of distilled water, and Sephadex G-2550 mM was added to this.
Add, stir, and leave for 30 minutes to 1 hour. Sephadex G-25 is removed by filtration or centrifugation, and the supernatant is concentrated and freeze-dried to obtain 90.3 mg of polysaccharide N9GI.

Example 1.2.3 When all polysaccharide N9GI was subjected to gel filtration with Sephadex G-100, polysaccharide NQGI was obtained.
It is confirmed that it contains only fractions.

That is, 160 mg of the above polysaccharide N9GI was dissolved in 20 m 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 N9GI a is obtained from both the first eluted fractions, and 61 mg of polysaccharide N9GI b from the second eluted fractions. It was confirmed by high performance chromatography and electrophoresis that these polysaccharides N9GIa and N9GIb were each a single compound.

(6) Specific Effects of the Invention As detailed above, the present invention provides an industrial method for producing polysaccharide N9GI.

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 tumor and Female A fibrosarcoma, and has very low toxicity, making it useful as an antitumor agent. 7 Yes. According to the present invention, a method for easily producing visible polysaccharide N9GI is provided. That is, the hot water extract of Melia azadirachta bark has a molecular weight range of 700 to 700.
The desired polysaccharide N9G I can be obtained with high purity simply by contacting it with a cross-linked dextran gel having a molecular weight of 5,000 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 drawings]

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. 8

Claims (4)

[Claims] (1) Melia azadirachta bark is extracted with hot water, the extract is purified by dialysis membrane method, alcohol precipitation method, or ultrafiltration method, and the obtained purified extract has a molecular weight cut-off range of 700 to 700. 5,000 with a lower limit of 100 or less, and collecting the polysaccharide from the contact aqueous solution. (a) 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)
. (c) Ultraviolet absorption spectrum measured in an aqueous solution 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 acisuron sulfuric acid reaction, and exhibits a bluish-green color when iodine is added. (2) The molecular weight cutoff range of the dextran gel is θ~7
Polysaccharide N9GI according to claim 1 which is 00
manufacturing method. (3) The molecular weight cutoff range of the dextran gel is θ~1
, 500, polysaccharide N9 according to claim 1, which is
GI manufacturing method. (4) The molecular weight cutoff range of the dextran gel is 100
5,000 to 5,000. The method for producing polysaccharide N9GI according to claim 1.