JPS6042329A - Production of polysaccharide n9gi - Google Patents

Abstract

PURPOSE:To produce the polysaccharide N9GI having remarkable immuno- activating antineoplastic activity, and useful as a low-toxic antineoplastic agent, by extracting the bark of Melia azadirachta with hot water, purifying the extract, and treating with a basic ion exchange material. CONSTITUTION:The bark of Melia azadirachta is extracted with hot water, purified by alcohol precipitation, dialysis and ultrafiltration, and made to contact with a basic ion exchange material [e.g. QAE Sephadex A-50 (product of Pharmacia Labs., Inc.)] to adsorb the impurities. The contact-treated aqueous solution is freeze-dried to obtain the objective polysaccharide N9GI having the following characteristics. Appearance, white-pale yellowish brown powder; solubility, soluble in water and insoluble in methanol, acetone, etc.; color reaction, positive to phenol-sulfuric acid raction, etc. The process can be carried out on a large scale with simple operation. The polysaccharide of the present invention has strong suppressing effect to the fixed solid cancer.

Description

DETAILED DESCRIPTION OF THE INVENTION 1. Background of the Invention Technical Field The present invention relates to a method for producing polysaccharide N 9 G 1.

In more detail, the present invention relates to a method for producing polysaccharide N9GI, which is characterized by purifying the hot water-extracted proboscis 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 is known that Melia azanolacta extracts have various pharmacological actions. That is, a method for obtaining skin cosmetics by extracting the bark, leaves, flowers, fruits, branches, root bark or ]01 fat of Melia azanolacta with water or a hydrophilic solvent, or by pulverizing it finely. (Special Public Interest Publication No. 52-28853.Sho 52-28853.
2-28854 and 53-10125), a method of extracting the original Melia azasolacta phase with a hydrophilic solvent and/or hot water to obtain a component having antibacterial activity and gastrointestinal/liver (beauty-improving effect) (Special Publication No. 53-10124)
A method has been reported (Japanese Patent Publication No. 53-13689) to obtain ingredients effective for the treatment of skin diseases and rheumatism by extracting the above-mentioned Melia azanolacta with a hydrophobic solvent.

1, the present invention 74 etc. was first introduced by Melia Azazolacta) No. 1
Alcohol is added to the hot water extract of the skin, and the resulting precipitate is collected (Japanese Unexamined Patent Publication No. 57-176914).
2.Hot Water Extraction', +11 is treated with a dialysis membrane, and the active ingredient is collected from the dialysate fluid (Japanese Patent Application Laid-Open No. 57-17691.5)
What extracts have antitumor effects? I reported that I was being fucked.

As a result of further research, the present inventors dissolved the above purified extract in water and mixed the aqueous solution with a molecular weight cut-off of approximately 1×10 to 1×
10 to 1 x 10 to 2 x 10 of a dull filter layer agent [e.g., Sephadex G100 (trade name, product of Pharma/A Co., Ltd.), Biokel 8 Lu p-100 (trade name, product of Ba-Rorad Co., Ltd.), etc.]. It was found that a novel polysaccharide N9GI can be obtained by filtering the polysaccharide using a gel and collecting the first two fractions of the three polysaccharide fractions. Although this method is excellent in obtaining highly pure polysaccharide N9G I, it is very difficult to implement on a large scale due to the complicated operation. However, it is not well suited for industrial implementation due to its lack of mechanical strength.

OBJECT 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 object of the present invention is to provide a method for producing polysaccharide N9Gl 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.

Extract Melia adsolacta bark with hot water, purify the extract by alcohol precipitation, barrel filtration, or ultrafiltration, and treat the resulting extracted and purified product with a base 1 bioion exchanger. A method for producing polysaccharide N9Gl having the following physicochemical properties.

・、B) Color and shape The dried product is white or light yellowish brown and powder.

←) Infrared decay (absorption spectrum K1--1 1Rν, nf, Xcwt, 3400, 2920, 16
30,1400゜1.360,11.50,1070,
1030, 920, 840 (c) Ultraviolet absorption absorption maximum when measured in 7g of water; shows only 5.1 nitrate absorption.

2) Solubility Soluble in water and insoluble in organic solvents such as tsukunol, ethanol, acetone, ether, chloroform, ethyl acetate, benzene and hexanoate.

(e) Color reaction It is positive for the phenol sulfuric acid reaction and the anthrone sulfuric acid reaction, and exhibits a blue-rimmed color when iodine is added.

3. Detailed Description of the Invention Melia azanolacta, the raw material plant for the method of the present invention, has a scientific name of Melia azadirachta, and grows naturally in tropical regions. It is a woody plant that can reach x or more. In the method of the present invention (#i, Ita 1 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, hot water is added to the shredded bark. Alternatively, it is carried out by adding water to the bark and stirring the mixture under heating.Heating can be carried out in a water bath or over an open flame. The time period is determined appropriately depending on the quality of the extract, 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 preferable to pre-extract the bark with an organic solvent and/or water at room temperature, and it is also desirable to remove unnecessary components in advance. Solvents used for extraction pretreatment include polar organic solvents such as methanol, ethanol, furopanol, pyridine, and acetone;
Examples include nonpolar organic solvents such as benzene, toluene, xyleno, n-hexane, chloroform, carbon tetrachloride, and ethyl acetate.

Since the hot water extract thus obtained still contains a large amount of impurities, it is purified by alcohol precipitation, dialysis membrane method, or ultrafiltration method. In the case of purification by alcohol precipitation, an alcohol such as methanol, ethanol, or fluorine is added to the 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 dipping in water, and the dialyzed solution is dried to a desired concentration or freeze-dried to obtain an extract.

The dialysis membrane has a molecular weight cut-off of 50,000 or less, such as Spectra der 1-6 (trade name, Svetatram.
Medical Industries, Inc.), Visking
Tube (trade name, Union Carbide product) is used. Alternatively, the molecular weight cut-off is 5,000 to IO,0
A hollow fiber type dialyzer of about 0.00 may also be used. For example, Terumo Corporation's Clearance T■-15 (
(trade name), Amicon's HI P5 (trade name, molecular weight cutoff 5,000) or HIPIO (trade name, molecular weight cutoff io, ooo) 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, an ultrafiltration membrane having a molecular weight cut-off of about 10,000 to 50,000 is used, and the purification is carried out under pressure according to a conventional method. The filtration membrane may be used as long as it has the above molecular weight cut-off.
Although there is no particular limitation, it is preferable to use a synthetic polymer cast into a nonwoven fabric or the like. An example of such a filtration membrane is Toyo Rudatsu Kogyo Co., Ltd. product (7) TSK-UF.
Membrane: TS-10 (molecular weight cut off to, 000), TS-3
'0 (30,000), TS-50 (50,000)
) and Amicon product ultrafiltration membranes: YMlo (molecular weight cut off 10,000), PMIO (same io, ooo),
YM 3. O (30,000), PM30 (30,000)
0,000) and XM50 (50,000). Particularly preferred is TS-50 (manufactured by Toyo Soda Kogyo Co., Ltd.). Pressure during filtration is approximately 0.1 to 2 kg/(:n
L2 is appropriate. Ultrafiltration simultaneously achieves concentration of hot water extract and removal of low molecular weight impurities. α of the concentrated liquid is 5 to 20 m97me, preferably 10 to 1.5 m9
/7ne.

The precipitate obtained by the alcohol precipitation method or the solid proboscis obtained by drying the dialyzed fluid in the dialysis method is dissolved in water and used as the purified extraction of the present invention.

The internal solution in the dialysis membrane method or ultrafiltration method is 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 strongly 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 aqueous extract solution and the basic ion exchanger are brought into contact by a patch method or a continuous method 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 lyophilized product is a white powder or a tan powder.

(b) Infrared absorption spectrum IRvKI3rcm-1: 3400, 2920, 16
30,1400゜aX 1360, 1150, 1070, 1030, 920.8
4.0 (c) Ultraviolet absorption spectrum When measured in an aqueous solution, it does not show an absorption maximum but only shows an unbroken absorption.

) Solubility Soluble in water and insoluble in organic solvents such as methanol, ethanol, acetone, ether, chloroform, ethyl acetate, benzene and hexane.

β Color reaction: Positive for phenol sulfuric acid reaction and anthrone sulfuric acid reaction, and exhibits blue-green color upon addition of iodine.

For reference, the polysaccharide N9Gl obtained above has a molecular weight cut-off of approximately ],
The polysaccharide is separated into two fractions by applying it to a column packed with a cell filtration agent No. 05 and eluting with distilled water.

The polysaccharide that elutes first is called polysaccharide N9Gla, and the polysaccharide that elutes later is called N9GIb. As the cell filtration agent, dextran gel, polyacrylamide gel, polyvinyl polymer gel, porous glass beads, etc. are used. These (for example, Sephadex G)
-200, Shichinoacrylic S-300 (product name, Farmana product, Sweden), Bioker 8ru p-: 3oo
(Product name, Biola, 1 company product, USA), Toyo ξ-
It is commercially available under the product name HW-60 (trade name, Toyo Rudatsu Kogyo Co., Ltd., Japan).

The structures and physicochemical properties of polysaccharide N9Gla and polysaccharide N9Glb are as follows.

Polysaccharide N9G l a a) Structure α-(1→・1)-glucan with arabinose in its main chain
A neutral polysaccharide with -(1→6) bonds and a composition ratio of glucose and arabinose of approximately 5:1.

mouth) color and shape The lyophilized product is a white powder.

c) Add methanol, ethanol, acetone,
Insoluble in organic solvents such as ether, chloroform, ethyl acetate, benzene, and hexanoate.

2) Color reaction When iodine is added to the phenol sulfuric acid reaction or the anthrone sulfuric acid reaction, a bluish-green color is produced.

e) Molecular weight Cefazo gives a single peak in 8-cell chromatography on a gas G-200 column, and the molecular weight is about 94,000.

) Specific rotation [α9, +], 36.00 (C=0.5,
11
Shows absorption only.

W) 13C nuclear magnetism / (Kikyo Ill spectrum in Shosui) 100 Mllz "C nuclear magnetism" measured using TMS (Tetrathyrun Run) l, 111
``ysubek) 1 is the following formula.

δppm: 02. +132.7. (i7.3,72
．． 9,74.8,78.1゜78.7,82.4,85
．． 5,99.2. ],0],,1. ．． IO8,9 1
'1''= (A' N (] G l b・i) :1
~ Blue α-(1→1)-glucan gold main chain, β-
(1→:3) via fucose, α-(1→0
) The composition ratio of arabinose, glucose, ambinose and fucose is 7', and the ratio is 5:2:1.

mouth) color and shape The lyophilized product is a white powder.

C) Soluble: Dissolve in water, methanol, ethanol, acetone,
Insoluble in organic solvents such as ether, coloform, ethyl acetate, benzene and hexano.

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 Sephadex G-200 column/Sephadex G-200 column shows a peak of 100%, and the molecular weight is about 21,000.

to) Specific rotation [α grandchild”: +1437° (C-205.■■20) 1.
) Infrared absorption spectrum Ultraviolet absorption spectrum When measured in an aqueous solution, it shows only the absorption maximum and shows only the terminal absorption.

li) 16C Nuclear Magnetic Resonance Spectrum The 100 MHz 13C nuclear magnetic resonance spectrum measured in heavy water using TMS (tetramethylsilane) as an external reference is as follows.

δpprn: 18.2, 62.3.62.7, 6
7.4.71.1, 72.0°73.2, 74.9,
78.3, 78.9, 82.9, 83.9°85.6
, 994.101.2 , 105.0 , 109.1 The polysaccharide N9GN of the present invention can be considered to be a mixture of the above-mentioned polysaccharide N9Gla and polysaccharide N9Glb, and as a result of pharmacological tests, it has been found that It was confirmed that it had a remarkable inhibitory effect on mouse transplanted tumors and female A solid type mouse transplanted tumors.

N9Gla and N9Glb also showed similar pharmacological activity.

Therefore, when used as an antitumor agent, the polysaccharide N9CI of the present invention may be further combined with polysaccharide N9G, Ia and polysaccharide N9G.
There is no need to separate it into lb and lb; in a mixture of both,
It is practical to use the polysaccharide N9Gl in the present invention.

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) A solution in which 05% 5% Calhoki/Methylcellulose CMC) was suspended in phosphate buffered saline (manufactured by Gibco, containing 9.5mMt of phosphorus; PBS). Both samples were dissolved to a predetermined concentration.

(Sarcoma 180 cancer cell transplantation) ■Sarcoma 180 cancer cells subcultured in the peritoneal cavity of CR mice were taken out with ascites and diluted appropriately with physiological saline to make the cell number 1.0 x 10"/me. 0.1 rne of this suspension was prepared for 4 year old, male IC.
Cells were transferred subcutaneously to the back of R mice using a syringe.

(Sample membrane force) Transfer Sarcoma 180 cancer cells (1σ from 6 whites)
The sample prepared above was intraperitoneally administered using a syringe once a day for 10 consecutive days. Six mice were used per 241 concentrations. The control is the above CMC person used as a solvent for the sample! JP PBS was administered in the same manner. The amount of Li applied was expressed as the number of mLi per kg of mouse body weight.

(Efficacy evaluation method) Cancer, I'ltl) Cancer tissue that had grown on the 21st day after 1 m transplantation was excised and its weight was measured (average value of 6 animals per group).

The effect amount was determined by taking the ratio (T/C) between this weight and that of the control. Control cancer tissue type is 15-3.5i9
It was. Invalidate the ratio value or 100 to 71% (-)
, 70-51% slightly effective (+), 50-21%
Those with diameter of 20 to 0φ are effective (/1), and those with 20 to 0φ are effective (/1).
0) Toshipuko. The results are shown in Table 1.

Table 1 Effect on Sarcoma 180 transplanted cancer (mouse) Ratio/Example Al 25. 56.1-1-.

5 '78.1 - 1 25 , 55.2 + 1 Comparative example B, s l 7□7 - ] 1 (Note) Comparative example A: Melia according to the method described in JP-A-57-17691.4・Hot water extract obtained by adding alcohol to the hot water extract of Azadirachta bark and collecting the resulting precipitate.

Comparative Example B: A hot water extract obtained by treating a hot water extract of Melia azanolacta bark with a dialysis membrane and collecting the active ingredient from the dialyzed solution according to the method described in JP-A-57-176915.

Test 1 Test Example 2 Effect on female A-fiber Aff sarcoma Female A-fiber subcultured in the peritoneal cavity of Ba1b/C mice (
(Meat 11iJi (MetllA f 1brosar
co+na)ill 1lfq fTake out together with ascites, dilute appropriately with physiological saline, and reduce the number of cells to 1.0X.
10 (I made the station so that it was solid/me). This j'
0.17111 f of lll cell suspension: 5 week old male Ba1
b/(The cells were transplanted subcutaneously on the back of the knee mouse using a syringe. - The number of transplanted cells per animal was 1.0 x 10''.

The sample was prepared in the same manner as in Test 1, Test Example 1, and administered intraperitoneally to mice 121 times on day 11 for 10 consecutive days.

Judgment was made by cutting out tumor tissue from the white of the 21st eye to which tumor cells had been implanted, and measuring its weight. The results are shown in Table 2.

Table 2 (Sho) Comparative 191JA and Comparative Example B are the same as in Table 1.

As is clear from Tables 1 and 2, the polysaccharide N9 of the present invention
CI is Zarco'-7180 solid gun and female AI
! aHas a strong inhibitory effect on fibrosarcoma. The potency was also about 5 to 6 times stronger than that of Comparative Examples A and B, indicating higher purity of the active ingredient.

Test Example 3 Acute Toxicity An acute toxicity test was conducted by administering the polysaccharide N9GI of the present invention to CR male mice weighing 20±11.
The value was 600 m97 kg or more when administered intraperitoneally.

As is clear from the results of the above-mentioned pharmacological tests, the polysaccharide N9GI of the present invention has remarkable antitumor properties and extremely low toxicity. Since it has a strong suppressive effect, the polysaccharide of the present invention is considered to have an immunostimulatory antitumor effect.

The polysaccharide of the present invention is effective against various cancer diseases,
The testing capacity varies depending on symptoms, age, weight, etc., but the usual dosage for adults is 100 to 2,500 mg per day.
It can be administered in 1 to 4 divided doses.

The polysaccharide N 9 G 1 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 are prepared using conventional excipients such as calcium carbonate, calcium phosphate, corn starch, potato starch, sugar, lactose, talc, magnesium stearate, etc. It may also contain gum arabic and the like. The 11-day body preparation for injection is water-based or oil-based, and is 70. No°
, an elixir or the like.

Injectable preparations (r'J, in the form of solutions or suspensions,
The clouding agent, stabilizer size/ζ may contain formulation agents such as dispersants, sterile distilled water, essential oils such as peanut oil, corn oil or non-aqueous solvents, polyethylene glycol, polypropylene glycol. It may be included.

For rectal administration i, H, it may be provided in the form of a suppository composition, which 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 m6 x 3) and methanol (50 g).
The resulting extraction residue l'Ik: was extracted three times with hot water at 200 rrt. The obtained extracts were combined and concentrated to dryness using a rotary evaporator to obtain a powder of 1960.5ff19.

(2) 1000 m9 of the powder obtained in (1) above was mixed with 2 ml of water.
Pure ethanol was gradually added to the resulting aqueous solution at room temperature while stirring, and when the ethanol concentration in the aqueous solution reached 80%, the addition was stopped, and the precipitate formed was collected by centrifugation. , 594.5 m2 of brown powder f: obtained.

(3) Add 500 mg of the powder obtained in (1) above to 50 mg of water.
This aqueous solution was poured into Spectra Pore 6 (molecular weight cut off: 50,000) and dialyzed against water.

The dialysis fluid was coagulated to dryness using a rotary evaporator and turned into a brown powder of 310m! I got li+.

(4) Powder 100 obtained in (2) or (3) above
mg was dissolved in 200 bottles of distilled water. 0.5 MN
QAE cefazo activated by aCL, Cus A-50 was mixed with the above aqueous solution, stirred for 6 hours, and then suction filtered. After dialyzing the filtrate, the desired polysaccharide N9G120.5m was lyophilized.
9 f: Obtained.

Example 2 Powder 100 obtained in Example 1 (2) or (3)
m9'f: Dissolved in 200 me distilled water. 0.5
M NaCt-activated QAE 10 Fade, Cus A-5
0 was packed in a column, the aqueous solution was passed through the column, the effluent was collected, dialyzed, concentrated, and directly dried to obtain 25.8 m9 of the desired polysaccharide N9Gl.

■1 Specific Effects of the Invention As detailed above, according to the present invention, the polysaccharide 9Gl
An industrial manufacturing method is provided.

As shown in the test example, polysaccharide N9GI
It exhibits strong inhibitory activity against 80% transplantation 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.

Patent applicant Terumo Corporation

Claims (1)

[Claims] (1) A purified extract obtained by extracting Melia azanolacta bark with hot water and refining the extract by alcohol precipitation, dialysis membrane method, or ultrafiltration farming method. A method for producing polysaccharide N9GI having the following physicochemical properties, which is characterized by treating N9GI with a basic/r-ion exchanger. (a) Color and Shape The chestnuts to be freeze-dried are white and pale yellow-brown powder. (b) Infrared 1 gland absorption spectrum I Rνmax cm -3400+ 2920.1
630 + 1400 +x36o, 1t5o, 1
070.1030.92o, s4° (c) Ultraviolet i (absorption spectrum measured in aqueous solution shows only terminal absorption without extremely thick absorption.) Solubility Soluble in water, methanol, ethanol, acetone, Insoluble in organic solvents such as ether, chloroform, ethyl acetate, benzene and hexa/etc. (e) Color reaction: It is positive for phenol sulfuric acid reaction and anomerone sulfuric acid reaction, and exhibits a bluish-green color when iodine is added.