JPS62185022A - Production of polysaccharide ma9 - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain a polysaccharide MA9 which is an antitumor agent, by extracting a tree bark of Melia azadirachta Linn. with hot water, purifying the resultant extract at a specific pH by an alcohol precipitation method and further purifying the precipitates by a gel filtration method. CONSTITUTION:A tree bark of Melia azadirachta Linn. is extracted with hot water and the resultant extract solution is adjusted to 1-3pH. An alcohol is then added to collect the formed precipitates, which are then dissolved in water. The above-mentioned aqueous solution is subjected to gel filtration and the initial fraction of trisected polysaccharide fractions is collected. The solvent is then removed to afford the aimed polysaccharide MA9. Methanol or ethanol is preferred for the alcohol to be added. The alcohol is preferably added in an amount to give 20-90%, particularly about 80% alcohol concentration. The purity of the product can be remarkably improved by simple operation to adjust the pH of the extract solution in the alcohol precipitation method.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improved method for producing polysaccharide MΔ9.

More specifically, the present invention extracts Melia azadirachta bark with hot water and purifies the extract by alcohol precipitation and gel filtration.
The present invention relates to a method for producing polysaccharide MΔ9 which exhibits tS of 1 to 3.

Polysaccharide MΔ9 is useful as an antitumor agent, and according to the method of the present invention, polysaccharide MA9 can be produced industrially and efficiently.

Tomoka JLJM The present inventors have previously found that the hot water extract of Melia azadirachta bark has an antitumor effect, and that the polysaccharide M△9 obtained by purifying the above extract has an even stronger antitumor effect. discovered this and proposed various extraction and purification methods (special
1 1977-38720, Mukai 57-48920, same 5
7-48921, 57-176914, same! )1-1
76915, 58-144320, 5B-144
321Jj and 58-144322). Furthermore, polysaccharide M
Δ9 is named polysaccharide N9GI in the above-mentioned patent publication, but both names are the same substance. According to the above-mentioned known method, polysaccharide MA9 is obtained by extracting the bark of Melia azadirachta with hot water, purifying the extract by, for example, alcohol precipitation, and further gel filtrating the obtained purified product, and dividing it into three parts. It is obtained by collecting the first two fractions from the bottom of the separated polysaccharide fraction.

However, the above $5! ! Method J does not necessarily take 11 minutes, and it is desired to develop a method that can obtain polysaccharide MA9 with higher purity.

(19) Purpose of the Invention The purpose of the present invention is to provide a manufacturing method suitable for industrial production of polysaccharide MA9.

In other words, an object of the present invention is to provide a method for purifying polysaccharide MA9 using simple operations without requiring special equipment.

The object of the present invention is to extract the bark of Melia azadirachta with hot water, and then subject the extract to alcohol precipitation.

In producing polysaccharide MA9 by purifying it by gel filtration method, the above alcohol precipitation method is carried out under the conditions of p111-3.

In general, the object of the present invention is achieved by carrying out the alcohol precipitation method described above using ethanol or methanol.

■0 Detailed Description of the Invention The method of the present invention involves extracting the bark of Melia azadirachta with hot water, adjusting the extract to pH 111 to 3, adding alcohol, collecting the generated precipitate, dissolving it in water, This is carried out by gel filtrating the aqueous solution, collecting the first two of the three polysaccharide fractions, and removing the solvent.

The scientific name of Melia azadirachta, the raw material plant, is Melia azadirachta.
Azadirachta Linnaeus (Heliaazadirach)
ta Linn, ) or Azadirachta indica 1ndica
Juss), which grows naturally in tropical areas and has a height of 101 cm.
It is a woody plant that reaches rL or higher. In the method of the invention, the bark of Melia azadirachta, preferably dried bark, is used as a raw material. Extraction is carried out according to conventional methods.

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. When the hot water extraction is carried out under a pressure of 1 to 3 atmospheres, the yield and yield of the product are improved, and the purity is also increased. The extraction time is determined appropriately according to the quality of the raw materials, etc., but it is usually 1 to 1 hour.
0 hour, preferably 2 to 3 hours, and repeated 2 to 5 times. 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 un)components. Solvents used for extraction pretreatment include polar solvents such as methanol, ethanol, propanol, pyridine, and acetone;
Examples include nonpolar organic solvents such as benzene, toluene, xylene, n-hexane, chloroform, carbon tetrachloride, and ethyl acetate.

Since the hot water extract thus obtained still contains many impurities, alcohol is added to the extract, and the resulting precipitate is collected and purified. The extract usually has a pH of 5.8, and alcohol can be added directly to it; however, the present inventors have found that when the pH of the extract is adjusted to 1 to 3 before adding alcohol, the IJtg level increases dramatically. I learned that I can improve myself.

Adjust the pH using m? Ii, 111kM, i of phosphoric acid, etc.
This is done by adding l&acid to the extract. Alcohols added to form a precipitate include methanol,
Ethanol is preferred, and it is desirable to add so that the alcohol concentration in the extract is 20 to 90%, particularly around 80%. The a: lees produced in the extract is collected by a conventional method, for example, by centrifugation. The collected precipitate was washed with an alcohol aqueous solution of the same concentration as above, and if desired,
Wash with near 00% alcohol and then with ether and dry.

In the method of the present invention, the hot water extract may be purified by a dialysis membrane method or an ultrafiltration method before being purified by an alcohol precipitation method. When purifying by the dialysis IgI method, the extract is placed in a dialysis membrane and dialyzed by soaking in water.
If desired, the dialyzed fluid is concentrated to dryness or lyophilized to obtain an extract. As a dialysis membrane, fractionated molecule ff15G,
Things with Goo on them, such as Su.

Vectra Boa 1-6 (product name, Subek 1-Lam Medical Industries Co., Ltd. product), Bisking Chi:
L-bu (trade name, manufactured by Union Carbide Company) is used. Alternatively, the molecular weight cut-off is 5.000 to 10,00
A fiber type dialyzer may be used.

For example, Chill Seven Co., Ltd. >J'Nproduct's Clearance rE-1
5 (Product Name), Amicon Rei's IIIP 5 (Product Name, Fractionated Molecules 115,000) Ma/', :Ha1ltP10
(fli product name, Fractionated Molecule Bio, ooo) can be used.

When purifying by ultrafiltration, the molecular weight cutoff is approximately 10,000.
It is carried out under pressure according to a conventional method using ultrafiltration membranes ranging from so to ooo. There are no particular restrictions on the material of the filtration membrane as long as it has a molecular weight cut-off as shown in E, but a membrane made by casting a synthetic polymer onto a non-woven fabric is preferably used. Examples of such filtration membranes include Toyo Dentatsu Kogyo (JAJ product [Sni-U[membrane: TS-10 (fraction molecule fi 110.000), TS-30 (fraction molecule fi 110.000)
0), TS・-50 (50,000) and Amicon "II product '7) 1 outside '5 over 1!: YHIG
(Fraction molecule 111G, Goo), PH10 (Fraction molecule 111G, Goo)
000), YH30 (30,000), PH30
(30,000) and (FXH!10 (50,000)
00).

Particularly desirable is TS-50 (manufactured by Toyo Notatsu T Gyosha).

Appropriate addition JH during filtration is approximately 0.1 to 2 Kg/an2. Ultrafiltration simultaneously achieves concentration of the hot water extract and removal of low molecular weight impurities. Concentration of concentrate is 5~
20#l! F/d, preferably 10-15 #J/-.

Dissolving the precipitate obtained by the alcohol precipitation method in water,
The aqueous solution is subjected to a molecular sieve treatment using a molecular sieve agent, preferably a gel filtration agent, with a molecular weight cut-off of about 1X103 to 1X105 to 1X103 to 2X105, and the bottom and first two fractions of the polysaccharide fraction divided into three are collected. , the desired polysaccharide MΔ9 is obtained by distilling off the solvent. As a gel filtration agent,
Dextran gel, polyacrylamide gel, polyvinyl polymer gel, porous glass beads, etc. are preferably used. These include Sephadex G-100,
It is commercially available under product names such as G-200 (Pharmacia product, Sweden), Biogel p-100 (Bio-Rad product, USA), and Toyovar 11-50 (Toyo Kaitako 2N1 product, Japan). The purified aqueous solution is passed through a column filled with these gel filtration agents, eluted with distilled water, and the first eluted polysaccharide fraction is collected and distilled to dryness or freeze-dried to obtain the desired polysaccharide MA9. .

The physicochemical properties of the polysaccharide MA9 thus obtained are consistent with those of the standard product, for example, the physicochemical properties of the polysaccharide N9GI described in JP-A-60-19717.

For reference, the polysaccharide obtained above was fractionated with approximately IX
The polysaccharide is fractionated into two fractions when applied to a column packed with 1G3 to 2X105 to 1X103 to 8X105 gel filtration agent and eluted with distilled water. The first eluted polysaccharide is MA9t1, and the later eluted polysaccharide is MΔ9b. As the gel filtration agent, dextran gel, polyacrylamide gel, polyvinyl polymer gel, porous glass beads, etc. are used. These include, for example, Sephadex G-200, Refryl 3-300 (trade name, Pharmacia?1 product, Sweden), Bioglu P-300 (trade name, Bio-Rad product, USA), [
・] Pearl +114. It is commercially available under product names such as -60 (trade name, Toyo Kaidat Kogyo Ne J'! 1 item, Japan).

The polysaccharide MA9 of the present invention can be considered to be a mixture of the above-mentioned polysaccharide MA9a and polysaccharide MA9b, and as a result of pharmacological tests, it was found that Sarcoma 180 solid mouse transplantation l. It was confirmed that the drug had a remarkable inhibitory effect on tumors and tumors transplanted into female A solid mice. In addition, the above polysaccharides MA9a and M
A9b also showed similar pharmacological activity.

Therefore, when using the polysaccharide MA9 of the present invention as an anti-M tumor agent, it is not necessary to further separate the polysaccharide MA9 of the present invention into polysaccharide MA9a and polysaccharide MA9b. It is practical to use it at MA9.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Melia azadirachta bark 1509 is soaked in 75% methanol 1.59% at room temperature for 5 hours. The leachate was filtered off, and 1.51 g of 75% methanol was added to the residue, and the mixture was heated at room temperature for 5 ml.
Soak for an hour. The leachate was filtered off, 1.5 liters of water was added to the residue, and the mixture was extracted by boiling for 3 hours. This hot water extraction operation is performed three times.

The hot aqueous extracts are collected and concentrated under reduced pressure to approximately 500 mL. Transfer the concentrated solution to TS and transfer it to Ichika membrane TS-50 (fractionated molecule Ffi 50).
．． Ultrafiltration is carried out using an ultrafiltration device FISC-60 (trade name, Toyo Soda J: Yorei Products) equipped with a filter (trade name, Toyo Soda J: Yorei Products).

After concentrating to about 100-150d at SEL,
150 to 200 me of distilled water is added to this, further ultrafiltration is performed, and the mixture is concentrated again to about 100 Id. Repeat this operation 5 to 10 times and get 1! ? Centrifuge the concentrated portion to remove insoluble matter. I: Clear liquid (solid content 9 #I!F/
m! ) and add hydrochloric acid to each portion to adjust the pH to 5.
0. 4.0°3.4. 3.0. 2.0. 1.0
Adjusted to, and! ! No added acid (pH 5
, 8) respectively.

Add 2 times m of ethanol to each solution (L ethanol final 67 m
%) or methanol 4 (final methanol concentration 80%)
) and collect the resulting precipitate. The precipitate is washed with ethanol or methanol and then dried. Each precipitate is subjected to gel filtration using Sephadex G-100, and the polysaccharide is collected from the first peak and freeze-dried to obtain polysaccharide MA9. The barhin 1 of MA9 in each sample (depending on the yield, purity, and total eluted polysaccharide) of polysaccharide MA9 was as shown in the table below.

From the above table, p during ethanol or methanol precipitation
H value is 3 or less, for example, pH is 2 during ethanol precipitation,
Alternatively, when the pH is set to 2.0 or 1.0 during methanol precipitation, the precipitate R is reduced compared to unadjusted or pH 4.0, but the percentage of MA9 in the total eluted polysaccharide is 90%.
As described above, the purity was so high that a purification step using Sephadex G100 was unnecessary.

Also of precipitation. 049G was as follows.

From the table above, the lower the pH value, the better. It is clear that the value of 049G is also lower.

(2) Specific Effects of the Invention According to the present invention, a method for producing polysaccharide MA9 with high purity is provided.

That is, in the method of the present invention, polysaccharide MA9 is obtained by extracting Melia a1F diracta bark with hot water and adjusting the extract to pH1.
・After adjusting to -3, alcohol is added, and the resulting precipitate is further removed by gel filtration to produce vA. According to the method of the present invention, the purity of the product can be dramatically increased by the extremely simple operation of adjusting the extract to p111-3 during the alcohol precipitation method.

Claims (3)

[Claims] (1) Melia azadirachta bark is extracted with hot water, and the extract is purified by alcohol precipitation and gel filtration to produce polysaccharide MA9, and the alcohol precipitation is carried out under conditions of pH 1 to 3. Polysaccharide MA characterized by
9 manufacturing methods. (2) The method for producing polysaccharide MA9 according to claim 1, wherein the alcohol precipitation method uses ethanol. (3) The method for producing polysaccharide MA9 according to claim 1, wherein the alcohol precipitation method uses methanol.