JPS6318961B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION Technical Field The present invention relates to a novel antitumor polysaccharide and a method for producing the same. More specifically, the present invention relates to an antitumor neutral polysaccharide obtained by purifying a hot water extract of neem bark, and a method for producing the same. The polysaccharide 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 a component having antibacterial effects and gastrointestinal/liver function improving effects (Japanese Patent Publication No. 53-10124); Method for obtaining ingredients effective for the treatment of skin diseases and rheumatism by extraction with hydrophobic solvents (Special Publication No. 13689, 1983)
has been reported. The present inventors previously discovered that a hot water extract of neem bark has cell division inhibiting activity, but as a result of further research, it was found that α-(1→ 4) A neutral polysaccharide in which arabinose is α-(1→6) linked to the main chain of glucan and the composition ratio of glucose and arabinose is approximately 5:1 was isolated. This polysaccharide is a new compound that has not been described in any literature and has excellent antitumor effects. OBJECT OF THE INVENTION Therefore, an object of the present invention is to provide the above polysaccharide useful as an anticancer agent. As described below, the polysaccharide of the present invention exhibits strong inhibitory activity against Sarcoma 180 ascites type and solid mouse transplanted tumors, and is particularly effective against established solid tumors. A further object of the present invention is to provide a method for producing the above polysaccharide. According to the method of the present invention, the polysaccharide is produced by treating a hot water extract of neem bark with a molecular sieve, particularly gel filtration, and collecting a compound having a molecular weight of about 94,000. DETAILED DESCRIPTION OF THE INVENTION The present invention first consists of an antitumor polysaccharide obtained from neem bark and having the following structure and properties. (a) Structure A neutral polysaccharide in which α-(1→6) arabinose is bonded 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 A single peak is obtained by column gel chromatography using a molecular sieve with a molecular weight cutoff of 1×10 ³ to 2×10 ⁵ , and the molecular weight is about 94,000. (f) Specific rotation [α] ²² _D : -35° (c=0.4, H ₂ O) (g) Infrared absorption spectrum As shown in Figure 1. IRν ^KBr _nax cm ^-1 : 3400, 2930, 1620 (H) Ultraviolet absorption spectrum When measured in an aqueous solution, it does not show an absorption maximum, but shows only terminal absorption. (li) ¹³ C nuclear magnetic resonance spectrum The 100MHz ¹³ C nuclear magnetic resonance spectrum measured in heavy water using TMS (tetramethylsilane) as an external standard is shown in Figure 2. δppm: 108.9, 101.1, 74.8, 72.9, 62.1 Second, the present invention provides a method for producing the above-mentioned antitumor polysaccharide, which comprises treating a hot water extract of neem bark with a molecular sieve and collecting a compound having a molecular weight of about 94,000. Consisting of Thirdly, the present invention provides the above-mentioned molecular sieve treatment to first reduce the molecular weight cut-off from about 1×10 ³ to 1×10 ⁵ to 1×10 ³ to 2×
10 ⁵ molecular sieve, and then the first polysaccharide fraction was further reduced to a molecular weight cut-off of approximately 1×10 ³ to 2×10 ^{5 .}
The above-mentioned method for producing an antitumor polysaccharide is performed by using a molecular sieve of 1×10 ³ to 8×10 ⁵ and collecting the first polysaccharide fraction. Fourthly, the present invention comprises a method for producing the antitumor polysaccharide, wherein the molecular sieve treatment is gel filtration, and the molecular sieve agent is a gel filtration agent. A fifth aspect of the present invention comprises a method for producing the antitumor polysaccharide, wherein the gelling agent is dextran gel, polyacrylamide gel, hydrophilic polyvinyl gel, or porous glass beads. When carrying out the method of the present invention, a hot water extract of neem bark is subjected to molecular sieve treatment, in particular gel filtration,
A compound with a molecular weight of approximately 94,000 is collected. Neem, which is a raw material plant for the method of the present invention, has a scientific name of Melia azadirachta, 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 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, it is carried out by adding hot water to shredded neem bark, or by adding water to neem 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 complete,
An extract is obtained by filtering the extraction mixture. 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 gel filtration 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 dialysis membrane method, put the extract into a dialysis membrane,
The dialyzed solution is dialyzed against water, and the dialyzed solution is concentrated to dryness or freeze-dried, as desired, to obtain an extract. Dialysis membranes with a molecular weight cutoff of 50,000 or less, such as Spectra Pore 1-6 (products of Spectrum Medical Industries), Visking
Tube (a product of Union Carbide) is used. 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. moreover,
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. Solvents used for extraction pretreatment include methanol, ethanol, propanol,
Examples include polar organic solvents such as pyridine and acetone, and non-polar organic solvents such as benzene, toluene, xylene, n-hexane, chloroform, carbon tetrachloride, and ethyl acetate. In the gel filtration step in the method of the present invention, the neem bark hot water extract is applied to a column packed with a gelling agent, and the eluted polysaccharide fraction has a molecular weight of approximately
It is carried out by collecting 94,000 polysaccharides using a conventional method. Gel filtration has a molecular weight cut-off of approximately 8×
It is carried out using up to 10 ⁵ gelling agents and the desired polysaccharide is collected from the first fraction. Examples of the gelling agent used in the above step include dextran gel, polyacrylamide gel, polyvinyl polymer gel, and porous glass beads. These include, for example, Cephadex G-100, G-200, Cephacryl S-300 (products of Pharmacia, Sweden), Biogel P
-100~P-300 (Biorad Inc., USA), Toyopearl HW-50, HW-55 (Toyo Soda Kogyo, Japan), CPG-10 (Electro Nucleonics, USA), etc. ing. Furthermore, in the method of the present invention, a desired polysaccharide can be isolated with high purity by using a combination of gelling agents having different molecular weight cutoffs. That is, the neem bark hot water extract has a molecular weight cut-off of approximately 1×
Distilled water was poured into a column packed with 10 ³ to 1 x 10 ⁵ to 1 x 10 ³ to 2 x 10 ⁵ gelling agents (e.g., Cephadex G-100, G-200, Biogel P-100, Toyopearl HW-50, etc.). When eluted with
It is fractionated into two fractions. The first eluted fraction is collected and mixed with a gel filter having a molecular weight cut-off of approximately 1×10 ³ to 2×10 ⁵ to 1×10 ³ to 8×10 ⁵ (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 first eluted fraction is collected and distilled to dryness or freeze-dried to obtain the desired antitumor polysaccharide. The polysaccharide thus obtained has the following structure and properties. (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 A single peak is obtained by column gel chromatography using a molecular sieve with a molecular weight cutoff of 1×10 ³ to 2×10 ⁵ , and the molecular weight is approximately 94,000. (f) Specific rotation [α] ²² _D : -35° (c=0.4, H ₂ O) (g) Infrared absorption spectrum As shown in Figure 1. IRν ^KBr _nax cm ^-1 : 3400, 2930, 1620 (h) Ultraviolet absorption spectrum When measured in an aqueous solution, it does not show an absorption maximum, but shows only terminal absorption. (li) ¹³ C nuclear magnetic resonance spectrum The 100MHz ¹³ C nuclear magnetic resonance spectrum measured in heavy water using TMS (tetramethylsilane) as an external standard is shown in Figure 2. δppm: 108.9, 101.1, 74.8, 72.9, 62.1 As a result of a pharmacological test, it was confirmed that the polysaccharide of the present invention has a remarkable inhibitory effect on Sarcoma 180 ascites type and solid mouse transplanted tumor. Next, a test example will be shown. Test example 1 Effect on Sarcoma 180 ascites cancer (sample preparation) Phosphate buffered saline (manufactured by Gibco, phosphoric acid
The sample was dissolved in a solution of 0.5% carboxymethylcellulose (CMC) suspended in PBS containing 9.5mM 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 (+), those with 40-11% are effective (),
10% to 0% is considered significant (). Table 1 shows the results.
Shown below. Test Example 2 Effect on Sarcoma 180 Solid Cancer (Sarcoma 180 Cancer Cell Transplantation) A cell suspension of 1.0×10 ⁸ cells/ml was prepared in the same manner as in Test Example. Add 0.1 ml of this suspension to a 4-week-old male
Cells were transplanted subcutaneously into the back of ICR mice using a syringe. (Efficacy evaluation method) 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 effect was determined by determining the ratio (T/C) between this weight and that of the control. Control cancer tissue weight is 3.0 ~
It was 4.5g. Ratio values of 100 to 71% are invalid (-), ratios of 70 to 51% are slightly valid (+), 50 to 21
% was considered effective (), and 20% to 0% was considered markedly effective (). The results are shown in Table 1. As is clear from Table 1, the polysaccharide of the present invention is
Sarcoma 180 has a particularly strong suppressive effect on ascites cancer among transplanted cancers.

[Table] Test Example 3 Effect on established solid tumors 1 x 10 ⁷ Sarcoma 180 cells were subcutaneously transplanted into the backs of male ICR mice (5 mice) and raised. The solid tumor was completely established and the size was about 1 to 2 g.10
A predetermined amount of the sample was intraperitoneally administered to the mouse once a day for 5 days. On the 21st day after transplantation, the tumor part was cut out and its weight was compared with that of the control group, and the efficacy was determined according to the method of Test Example 2. The results are shown in Table 2.

[Table] Test Example 4 Acute Toxicity An acute toxicity test was conducted by administering the polysaccharide of the present invention to ICR male mice weighing 20±1 g. As a result, LD ₅₀
The values were 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 polysaccharide 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 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, and the dosage varies depending on symptoms, age, body weight, etc., but is usually 100 to 2,500 doses per day for adults.
mg, and can be administered in 1 to 4 divided doses. The polysaccharides of the present invention are formulated for oral or parenteral administration using any required pharmaceutical carriers or excipients. Tablets, powders, capsules, granules, etc. for oral administration contain conventional excipients such as calcium carbonate, calcium phosphate, corn starch, potato starch, sugar, lactose, talc, magnesium stearate, gum arabic, etc. Good too. Liquid preparations for oral administration may be aqueous or oily suspensions, solutions, syrups, elixirs, and the like. Injectable preparations are in the form of solutions or suspensions;
It may contain formulation agents such as suspending, stabilizing or dispersing agents, sterile distilled water, essential oils such as peanut oil, corn oil or non-aqueous solvents;
It may contain polyethylene glycol, polypropylene glycol, etc. For rectal administration, they may be provided in the form of suppository compositions and may contain well-known pharmaceutical carriers such as polyethylene glycol, lanolin, coconut oil, and the like. Next, the present invention will be explained in more detail with reference to 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 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. 273 mg of polysaccharide from the first eluting fraction
is obtained. Next, 50 mg of this solution was dissolved in 5 ml of distilled water, poured into a column (diameter 4.0 cm, length 50.0 cm) packed with Cephadex G-200, 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 two. 18 mg of the desired antitumor polysaccharide was obtained from the first eluted polysaccharide fraction. This compound was confirmed to be a single compound by high performance liquid chromatography and electrophoresis. In addition, in the above gel filtration, Cephadex G
Similar results were obtained when Cephacryl S-300 was used instead of -200. Specific Effects of the Invention As detailed above, according to the present invention, firstly,
Anti-tumor polysaccharides are provided. This polysaccharide is a new compound that has not been described in any literature, and as shown in the test examples, it shows strong inhibitory activity against Sarcoma 180 ascites type and solid mouse transplanted tumors, etc., and has very low toxicity, so it can be used as an anticancer agent. It is useful as According to the present invention, secondly, a method for producing the above polysaccharide is provided. That is, the polysaccharide is obtained by treating a hot water extract of neem bark with a molecular sieve and collecting a compound having a molecular weight of about 94,000. According to the present invention, thirdly, an advantageous method for producing the above polysaccharide is provided. That is, the polysaccharide undergoes the above molecular sieve treatment to have a molecular weight cut-off of approximately 1×10 ³ to 1×10 ⁵ to 1
×10 ³ to 2 × 10 ⁵ molecular sieve and molecular weight cutoff approximately 1 ×
High purity can be obtained by carrying out the process in combination with a molecular sieve of 10 ³ to 2×10 ⁵ to 8×10 ⁵ . According to the present invention, fourthly, a more advantageous method for producing the above polysaccharide is provided. That is, the polysaccharide is
It can be obtained more easily by performing gel filtration using a gel filtration agent as the molecular sieve treatment. According to the present invention, fifthly, a more advantageous method for producing the above polysaccharide is provided. That is, the polysaccharide is
High purity can be easily obtained by using dextran gel, polyacrylamide gel, hydrophilic polyvinyl gel or porous glass beads as a gelling agent.

[Brief explanation of the drawing]

FIG. 1 shows the infrared absorption spectrum of the antitumor polysaccharide of the present invention, and FIG. 2 shows the ¹³ C nuclear magnetic resonance spectrum of the same substance.

Claims (1)

[Scope of Claims] 1. An antitumor polysaccharide obtained from neem bark and having the following structure and properties. (a) Structure A neutral polysaccharide in which α-(1→6) arabinose is bonded 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 A single peak is obtained by column gel chromatography using a molecular sieve with a molecular weight cutoff of 1×10 ³ to 2×10 ⁵ , and the molecular weight is about 94,000. (f) Specific rotation [α] ²² _D : -35° (c=0.4, H ₂ O) (g) Infrared absorption spectrum As shown in Figure 1. IRν ^KBr _nax cm ^-1 : 3400, 2930, 1620 (H) Ultraviolet absorption spectrum When measured in an aqueous solution, it does not show an absorption maximum, but shows only terminal absorption. (li) ¹³ C nuclear magnetic resonance spectrum The 100MHz ¹³ C nuclear magnetic resonance spectrum measured in heavy water using TMS (tetramethylsilane) as an external standard is shown in Figure 2. δppm: 108.9, 101.1, 74.8, 72.9, 62.1 2. An antitumor polysaccharide having the following structure and characteristics characterized by treating a hot water extract of neem bark with a molecular sieve and collecting a compound having a molecular weight of about 94,000. Manufacturing method. (a) Structure A neutral polysaccharide in which α-(1→6) arabinose is bonded 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 A single peak is obtained by column gel chromatography using a molecular sieve with a molecular weight cutoff of 1×10 ³ to 2×10 ⁵ , and the molecular weight is about 94,000. (f) Specific rotation [α] ²² _D : -35° (c=0.4, H ₂ O) (g) Infrared absorption spectrum As shown in Figure 1. IRν ^KBr _nax cm ^-1 : 3400, 2930, 1620 (H) Ultraviolet absorption spectrum When measured in an aqueous solution, it does not show an absorption maximum, but shows only terminal absorption. (li) ¹³ C nuclear magnetic resonance spectrum The 100MHz ¹³ C nuclear magnetic resonance spectrum measured in heavy water using TMS (tetramethylsilane) as an external standard is shown in Figure 2. δppm: 108.9, 101.1, 74.8, 72.9, 62.1 3 The above molecular sieve treatment first
10 ³ -1×10 ⁵ to 1×10 ⁵ to 2×10 ⁵ molecular sieves, and then the first polysaccharide fraction is further divided into molecular weight fractions of about 1×10 ³ to 2×10 ⁵ to 1× 10 ³ ~ 8
3. The method for producing an antitumor polysaccharide according to claim 2, which is carried out using a molecular sieve of ×10 ⁵ and collecting the first polysaccharide fraction. 4. The method for producing an antitumor polysaccharide according to claim 2 or 3, wherein the molecular sieving treatment is gel filtration, and the molecular sieving agent is a gel filtration agent. 5. Claim 4, wherein the gelling agent is dextran gel, polyacrylamide gel, hydrophilic polyvinyl gel, or porous glass beads.
The method for producing the antitumor polysaccharide described in Section 1.