JPS6153336B2 - - Google Patents

Description

BACKGROUND OF THE INVENTION Technical Field The present invention relates to an antitumor purified neem bark extract and a method for producing the same. More specifically, the present invention relates to an antitumor purified neem bark extract obtained by purifying a hot water extract of neem bark by a specific method, and a method for producing the same. The extracted and purified product of the present invention is useful as a therapeutic agent for cancer. Prior Art It has been known that neem extracts have various pharmacological actions. Namely, neem bark, leaves,
A method for obtaining skin cosmetics by extracting flowers, fruits, branches, root bark, or resin with water or a hydrophilic solvent, or by pulverizing them (Japanese Patent Publications No. 52-28853, No. 52-28854)
and 53-10125), a method for extracting the above neem raw material with a hydrophilic solvent and/or hot water to obtain components having antibacterial effects and gastrointestinal/liver function improving effects (Special Publication No. 53-10124), and the above neem raw material A method for obtaining ingredients effective for the treatment of skin diseases and rheumatoid arthritis by extracting them with a hydrophobic solvent (Special Publication No. 53-13689)
has been reported. The present inventors previously discovered that a hot water extract of neem bark has an antitumor effect, but as a result of further research, a purified product obtained by processing the hot water extract of neem bark using a specific method was found. The present invention was completed after discovering that it has a stronger antitumor effect. OBJECT OF THE INVENTION Accordingly, an object of the present invention is to provide a purified neem bark extract useful as an anticancer agent. The extracted and purified product of the present invention is a novel substance, and as described below, it exhibits strong inhibitory activity against Sarcoma 180 ascites type and solid mouse transplanted tumors and female A solid mouse transplanted tumors, and particularly against established solid tumors. It is shown to be effective. A further object of the present invention is to provide a method for producing the purified neem bark extract. DETAILED DESCRIPTION OF THE INVENTION The present invention first involves extracting neem bark with hot water;
The extract is purified by alcohol precipitation method or dialysis membrane method, the obtained purified product is dissolved in water, and the aqueous solution has a molecular weight cut-off of about 1×10 ³ to 1×10 ⁵ to 1×10 ³
Treated with molecular sieve using ~2×10 ⁵ molecular sieve agent,
This is a purified antitumor neem bark extract obtained by collecting the first fraction of the polysaccharide fractions. This extracted and purified product has the following physicochemical properties. (b) Color and shape The freeze-dried product is a white powder or a pale yellowish brown powder. (b) Infrared absorption spectrum As shown in Figure 1 (extracted and purified product in Example 1). IRν ^KBr _nax cm ^-1 : 3400, 1630, 1030 (c) Ultraviolet absorption spectrum When measured in an aqueous solution, it shows no absorption maximum, but only terminal absorption. (d) Solubility Soluble in water and insoluble in organic solvents such as methanol, ethanol, acetone, ether, chloroform, ethyl acetate, benzene and hexane. (e) Color reaction It is positive for the phenol sulfuric acid reaction and the anthrone sulfuric acid reaction, and exhibits a bluish-green color when iodine is added. Second, the present invention extracts neem bark with hot water,
The extract is purified by alcohol precipitation method or dialysis membrane method, the obtained purified product is dissolved in water, and the aqueous solution has a molecular weight cut-off of about 1×10 ³ to 1×10 ⁵ to 1×10 ³
Treated with molecular sieve using ~2×10 ⁵ molecular sieve agent,
This is a method for producing a purified antitumor neem bark extract having the above-mentioned physicochemical properties obtained by collecting the first fraction among the polysaccharide fractions. A third aspect of the present invention is the method for producing the purified neem bark extract, wherein the molecular sieving treatment is gel filtration, and the molecular sieving agent is a gel filtration agent. A fourth aspect of the present invention is the method for producing the purified neem bark extract, wherein the gelling agent is dextran gel, polyacrylamide gel, hydrophilic polyvinyl gel, or porous glass beads. The scientific name of neem, which is the raw material plant for the method of the present invention, is Melia azadirachta Linnaeus.
Azadirachta Linn.) or Azadirachta indica Juss is a woody plant that grows over 10 meters in height and grows naturally in tropical regions. In the method of the present invention, a hot water extract of neem bark is used as a raw material. The extraction treatment of neem bark with hot water is carried out according to a conventional method. That is, adding hot water to shredded neem bark or adding water to neem bark;
This is carried out by 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, the extract mixture is filtered to obtain an extract. Since the neem bark hot water extract thus obtained contains a large amount of impurities, it is desirable to purify the extract by alcohol precipitation or dialysis membrane method before subjecting it to the molecular sieve treatment step of the present invention. . For example, when purifying by alcohol precipitation, an alcohol such as methanol or ethanol 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.
As the dialysis membrane, those having a molecular weight cut-off of 50,000 or less are used, such as Spectra Pore 1 to 6 (products of Spectrum Medical Industries) and Visking Tube (products of Union Carbide). The extract obtained by purification by alcohol precipitation method or dialysis membrane method is dissolved in water to obtain a hot water extract of neem bark, which is a raw material for the method of the present invention. Furthermore, prior to the hot water extraction treatment of neem bark, it is also desirable to pre-extract neem bark with an organic solvent and/or water at room temperature to remove unnecessary components in advance. Solvents used for extraction pretreatment include polar organic solvents such as methanol, ethanol, propanol, pyridine, and acetone;
Benzene, toluene, xylene, n-hexane,
Examples include nonpolar organic solvents such as chloroform, carbon tetrachloride, and ethyl acetate. The molecular sieve treatment in the method of the present invention is preferably carried out by gel filtration using a gel filtration agent. The gelling agent has a molecular weight cut-off of approximately 1×10 ³ to 1×10 ⁵ to 1×10 ³ to 2×10 ⁵
The following are used: dextran gel, polyacrylamide gel, polyvinyl polymer gel,
Porous glass beads and the like are preferably used. These include, for example, Cephadex G-100, G-200 (Pharmacia product, Sweden), Biogel P
-100 (BioRad product, USA), Toyo Pearl
It is commercially available under product names such as HW-50 (manufactured by Toyo Soda Kogyo Co., Ltd., Japan). When the above-described hot water extract of neem bark is passed through a column filled with these gelling agents and eluted with distilled water, the polysaccharide is separated into three fractions. The first eluted fractions are collected and distilled to dryness or freeze-dried to obtain the desired extracted and purified product. For reference, the extracted and purified product obtained above was further analyzed with a molecular weight cut-off of approximately 1×10 ³ to 2×10 ⁵ to 1×10 ³
~8×10 ⁵ gelling agents (e.g. Cephadex G-200, Cephacryl S-300, Biogel P-
300, Toyopearl HW-60, etc.) and eluted with distilled water, the polysaccharide is further fractionated into two fractions. The fraction eluted first is called polysaccharide A, and the fraction eluted later is called polysaccharide B. These polysaccharides have the following structure and properties. Polysaccharide A (a) Structure A neutral polysaccharide in which arabinose is α-(1→6) linked to the main chain of α-(1→4)-glucan, and the composition ratio of glucose and arabinose is approximately 5:1. (b) Color and shape The freeze-dried product is a white powder. (c) Solubility Soluble in water, methanol, ethanol, acetone, ether, chloroform, ethyl acetate,
Insoluble in organic solvents such as benzene and hexane. (d) Color reaction It is positive for the phenol sulfuric acid reaction and the anthrone sulfuric acid reaction, and exhibits a bluish-green color when iodine is added. (e) Molecular weight It gives a single peak in Sephadex G-200 column gel chromatography, and the molecular weight is approx.
It is 94000. (F) Infrared absorption spectrum As shown in Figure 2. IRν ^KBr _nax cm ^-1 : 3400, 2930, 1620 (g) Ultraviolet absorption spectrum When measured in an aqueous solution, it shows no absorption maximum, but only terminal absorption. (H) ¹³ C nuclear magnetic resonance spectrum The 100 MHz ¹³ C nuclear magnetic resonance spectrum measured in heavy water using TMS (tetramethylsilane) as an external standard is shown in Figure 3. Polysaccharide B (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. IRν ^KBr _nax cm ^-1 : 3400, 2930, 1630 (g) Ultraviolet absorption spectrum When measured in an aqueous solution, it does not show an absorption maximum, but shows only terminal absorption. (H) ¹³ C nuclear magnetic resonance spectrum The 100 MHz ¹³ C nuclear magnetic resonance spectrum measured in heavy water using TMS (tetramethylsilane) as an external standard is shown in Figure 5. The extracted and purified product of the present invention can be considered to be a mixture of polysaccharide A and polysaccharide B, and as a result of pharmacological tests, it has been shown to be significantly effective against Sarcoma 180 ascites type and solid mouse transplanted tumor and female A solid mouse transplant tumor. It was confirmed that it has a strong inhibitory effect. Moreover, the above-mentioned polysaccharides A and B also showed similar pharmacological activity. Therefore, when used as an anticancer agent, it is not necessary to further separate the purified extracted product of the present invention into polysaccharide A and polysaccharide B, and the purified extracted product of the present invention is used in the state of a mixture of both, that is, in the state of the purified extracted product of the present invention. It is practical to use it in Next, test examples of the extracted and purified product of the present invention will be shown. Test example 1 Effect on Sarcoma 180 ascites cancer (sample preparation) Phosphate buffered saline (manufactured by Gibco, phosphoric acid 9.5m
The sample was dissolved in a solution of 0.5% carboxymethyl cellulose (CMC) suspended in PBS) 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
It was transplanted into the abdominal cavity of a week-old male ICR mouse using a syringe. Therefore, the number of transplanted cells per animal was 1.0×10 ⁷ cells. (Sample Administration) Starting from the next day after transplanting the Sarcoma 180 cancer cells, 0.1 ml of the sample prepared above was administered once a day for 4 consecutive days using a syringe into the abdominal cavity. Six mice were used per sample per concentration. As a control, the above-mentioned PBS containing CMC, 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 efficacy) The weight of each mouse was measured on the 7th day after cancer cell transplantation. Next, the weight of the mouse was measured after removing the entire amount of ascites that had accumulated in the abdominal cavity. The difference in body weight before and after ascites collection is considered the amount of ascites. The collected ascitic fluid was sucked into a hematocrit tube and centrifuged at low temperature using a hematocrit measuring rotor to obtain an acytocrit value, which corresponds to the hematocrit value of blood (percentage of cancer cells in ascites).
Multiplying the amount of ascites by this value gives the volume of cells in the total ascites. This is defined as the total cell volume (TPCV). In controls, the total ascitic fluid volume was 6-10 ml, and the TPCV was 1.6-2.5.
It became ml. TPCV of sample-treated mice and control-treated mice
Taking the TPCV ratio (T/C), those with 100-66% are not effective against cancer (-), those with 65-41% are somewhat effective (+), and those with 40-11% are effective (〓) ,
10% to 0% is considered to be markedly effective (〓). Table 1 shows the results.
Shown below.

[Table] Test Example 2 Effect on Sarcoma 180 Solid Cancer (Transplantation of Sarcoma 180 Cancer Cells) A cell suspension of 1.0×10 ⁸ cells/ml was prepared in the same manner as in Test Example 1. 0.1ml of this suspension at 4 weeks of age
Cells were transplanted subcutaneously into the back of male ICR mice using a syringe. (Efficacy evaluation method) (1) Cancer tissue that had grown on the 21st day after cancer cell transplantation was removed, and its weight was measured (average value of 6 animals per group). The ratio of this weight to that of the control (T/C)
The effectiveness was determined based on the results. The control cancer tissue weight was 3.0-4.5 g. Ratio value is 100-71%
Disabled (-), 70-51% somewhat effective (+), 50-21% effective (〓), 20-0
% was marked as a significant effect (〓). The results are shown in Table 2.

[Table] (2) The above judgment method (1) is exactly the same as the above judgment method (1) except that the cancer tissue is removed on the 35th day after cancer cell transplantation instead of on the 21st day. The effectiveness was determined using the following method. The results are shown in Table 3.

[Table] As is clear from Tables 1 to 3, the extracted and purified product of the present invention has a strong inhibitory effect on Sarcoma 180 transplanted cancer. Test Example 3 Effect on established solid tumors (1) 1 x 10 ⁷ Sarcoma 180 cells were subcutaneously transplanted into the backs of male ICR mice (5 mice) and raised. A predetermined amount of the sample was intraperitoneally administered to the mouse once a day for 5 days from day 10, when the solid tumor had completely established and reached a size of about 1 to 2 g. On the 21st day after transplantation, the tumor area was cut out and its weight was compared with that of the control group.
Effects were determined according to the method of Test Example 2. The results are shown in Table 4.

[Table] (2) In the above method, the effect on established solid cancer was measured in exactly the same manner as the above method except that the tumor was removed on the 35th day after transplantation instead of on the 21st day. The results are shown in Table 5.

[Table] Test Example 4 Effect of oral administration on Sarcoma 180 solid cancer Sarcoma 180 under the same conditions as Test Examples 2 and 3
The effect of oral administration of the purified extract of the present invention on 180 solid cancer was measured. However, the number of transplanted cells was 2×10 ⁶ cells/mouse, and the determination was made by weight measurement on the 32nd day. In addition, the sample was forcibly administered into the stomach using a sonde at a rate of 0.1 ml/time/mouse at a predetermined concentration. The results are shown in Table 6.

[Table] From Table 6, it is clear that oral administration also has an antitumor effect at 250 mg/Kg. Test Example 5 Effect on female A fibrosarcoma Female A subcultured in the peritoneal cavity of Balb/C mice
Fibrosarcoma (Meth A fibrosarcoma) cells were removed together with ascites, diluted appropriately with physiological saline,
The number of cells was adjusted to 1.0×10 ⁷ 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 ⁶ cells. Samples were prepared in the same manner as in Test Examples 2 and 3, and administered intraperitoneally on the designated administration day. 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 7.

[Table] As shown in Table 7, even for female A fibrosarcoma, which is a syngeneic tumor, the extracted and purified product of the present invention
mg/Kg, 8 to 10 doses significantly inhibit tumor growth. Test Example 6 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 for intraperitoneal administration and 1000 mg/Kg or more for oral administration. As is clear from the results of the pharmacological tests mentioned above,
The extracted and purified product of the present invention has remarkable antitumor properties and extremely low toxicity, so it has excellent properties as an anticancer agent. Furthermore, since it has a particularly strong suppressive effect on established solid cancers, the extracted and purified product of the present invention is considered to have an immunostimulatory antitumor effect. The extracted and purified product of the present invention is effective against various cancer diseases, and the dosage varies depending on the symptoms, age, body weight, etc., but is usually 100 to 100 mg per day for adults.
The dose is 2500 mg and can be administered in 1 to 4 doses. The extracted and purified product of the present invention can be formulated for oral or parenteral administration using any necessary 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 Reference Examples and Examples. Reference example Production of neem bark hot water extract (1) 50g of dried neem bark was mixed with benzene (500ml x
3) and methanol (500 ml x 3) at room temperature for 24 hours, and the resulting extraction residue was extracted three times with 200 ml of hot water. The obtained extracts were combined and concentrated to dryness using a rotary evaporator to obtain 1960.5 mg of powder. (2) Dissolve 1000 mg of the powder obtained in (1) above in 200 ml of water, gradually add pure ethanol to the resulting aqueous solution at room temperature while stirring, and when the ethanol concentration in the aqueous solution reaches 80%. The addition was stopped, and the formed precipitate was collected by centrifugation to obtain 594.5 mg of brown powder. (3) 500 mg of the powder obtained in (1) above was dissolved in 50 ml of water, and this aqueous solution was poured into Spectra Pore 6 (molecular weight cut off: 50,000) and dialyzed against water. The dialyzed fluid was concentrated to dryness using a rotary evaporator to obtain 310 mg of brown powder. Example 1 1020 mg of the neem bark hot water extract obtained in Reference Example (2) or (3) above was dissolved in 20 ml of distilled water, and a column (diameter 7.0 cm,
(length 35.0 cm) and gel filtration was performed using distilled water. When gel filtration is performed while quantifying the sugar content in the eluate using the phenol-sulfuric acid method, the polysaccharide is fractionated into three parts. The solvent was distilled off from the first eluted fraction to obtain 273 mg of the desired purified neem bark extract. For reference, 50 mg of the above extracted and purified product was dissolved in 5 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 was performed while quantifying the sugar content in the eluate using the phenol-sulfuric acid method, the polysaccharide was fractionated into two. 18 mg of polysaccharide A was obtained from the first eluted fraction, and 16 mg of polysaccharide B was obtained from the second eluted fraction. The above polysaccharides A and B
was confirmed to be a single compound by high-performance liquid chromatography and electrophoresis. In Example 1 of the method of the present invention, Biogel P-
Similar results were obtained using 100. Specific Effects of the Invention As detailed above, the present invention provides, firstly, an antitumor purified neem bark extract. This extracted and purified product 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 ascites type and solid mouse transplanted tumors, female A solid mouse transplanted tumors, etc., and is toxic. It is useful as an anticancer agent because it has a very low value. According to the present invention, secondly, a method for producing the above-mentioned extracted and purified product is provided. That is, the extracted and purified product is obtained by extracting neem bark with hot water, purifying the extract 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
~1×10 ⁵ to 1×10 ³ to 2×10 ⁵ molecular sieve treatment using a molecular sieving agent, and among the three polysaccharide fractions, the first fraction is collected. It will be done. Thirdly, the present invention provides an advantageous method for producing the extracted and purified product. That is, there is provided a method for producing the extracted and purified product, wherein the molecular sieve treatment is gel filtration, and the molecular sieve is a gel filtration agent. Fourthly, the present invention provides a more advantageous method for producing the extracted and purified product. That is, there is provided a method for producing the extracted and purified product, wherein the gelling agent is dextran gel, polyacrylamide gel, hydrophilic polyvinyl gel, or porous glass beads.

[Brief explanation of the drawing]

FIG. 1 shows the infrared absorption spectrum of the purified antitumor neem bark extract of the present invention (purified product of Example 1). Figure 2 shows the infrared absorption spectrum of polysaccharide A, and Figure 3 shows the ¹³ C nuclear magnetic resonance spectrum of the same substance. Figure 4 shows the infrared absorption spectrum of polysaccharide B, and Figure 5 shows ^{the 13} C nuclear magnetic resonance spectrum of the same substance.

Claims (1)

[Claims] 1. Neem bark is extracted with hot water, the extract is purified by alcohol precipitation method or dialysis membrane method, the obtained purified product is dissolved in water, and the aqueous solution has a molecular weight cut-off of about 1. ×10 ³ to 1 × 10 ⁵ to 1 × 10 ³ to 2 × 10 ⁵ molecular sieve treatment using a molecular sieving agent, and among the three polysaccharide fractions, the first fraction is collected. Antitumor purified neem bark extract. This extracted and purified product 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 (extracted and purified product in Example 1). IRν ^KBr _nax cm ^-1 : 3400, 1630, 1030 (c) Ultraviolet absorption spectrum When measured in an aqueous solution, it shows no absorption maximum but only terminal absorption. (d) Solubility Soluble in water and insoluble in organic solvents such as methanol, ethanol, acetone, ether, chloroform, ethyl acetate, benzene and hexane. (e) Color reaction It is positive for the phenol sulfuric acid reaction and the anthrone sulfuric acid reaction, and exhibits a bluish-green color when iodine is added. 2. Neem bark is extracted with hot water, the extract is purified by alcohol precipitation method or dialysis membrane method, the obtained purified product is dissolved in water, and the aqueous solution has a molecular weight cut-off of about 1×10 ³ to 1× 10 ⁵ to 1 x 10 ³ to 2 x 10 ⁵ molecular sieve treatment using a molecular sieving agent, and the following physicochemical properties obtained by collecting the first fraction among the three polysaccharide fractions. A method for producing a purified antitumor neem bark extract having antitumor properties. (b) Color and shape The freeze-dried product is a white powder or a pale yellowish brown powder. (b) Infrared absorption spectrum As shown in Figure 1 (extracted and purified product in Example 1). IRν ^KBr _nax cm ^-1 : 3400, 1630, 1030 (c) Ultraviolet absorption spectrum When measured in an aqueous solution, it shows no absorption maximum, but only terminal absorption. (d) Solubility Soluble in water and insoluble in organic solvents such as methanol, ethanol, acetone, ether, chloroform, ethyl acetate, benzene and hexane. (e) Color reaction It is positive for the phenol sulfuric acid reaction and the anthrone sulfuric acid reaction, and exhibits a bluish-green color when iodine is added. 3. The method for producing an antitumor purified neem bark extract according to claim 2, wherein the molecular sieving treatment is gel filtration, and the molecular sieving agent is a gel filtration agent. 4. Claim 3, wherein the gelling agent is dextran gel, polyacrylamide gel, hydrophilic polyvinyl gel, or porous glass beads.
A method for producing a purified antitumor neem bark extract as described in 2.