JPS6152809B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the Invention The present invention relates to a novel neem bark extract with anti-neoplastic activity. It is known that neem contains components with pharmacological effects, and to date, neem has been found to contain components that improve skin function, components that have antibacterial effects, components that improve gastrointestinal and liver functions, and anti-rheumatic components. The active ingredients are known (Tokukosho
52-28853, 52-28854, 53-10124, 53-
10125, 53-13689). In addition, the present inventors previously discovered that a neem axilla extract obtained by extracting neem bark with hot water and concentrating the extract to dryness has cell division inhibiting activity, but further research As a result of repeated research, I learned that by adding alcohol to a hot water extract of neem bark and collecting the resulting precipitate, a neem bark extract with higher activity against malignant neoplasms could be obtained.
The invention has been completed. OBJECTS OF THE INVENTION It is therefore an object of the present invention to provide a neem bark extract having high activity against malignant neoplasms. DETAILED DESCRIPTION OF THE INVENTION The present invention has, firstly, an anti-neoplastic effect obtained by extracting neem bark with hot water, adding alcohol to the resulting extract, and collecting the resulting precipitate. Provides neem bark extract. Second, the present invention is obtained by pre-treating neem bark with water at room temperature, extracting the resulting material to be treated with hot water, adding alcohol to the extract, and collecting the resulting precipitate. The present invention provides a neem bark extract having anti-neoplastic activity. The third aspect of the present invention is to pre-treat neem bark sequentially with a polar organic solvent and water at room temperature, extract the resulting treated material with hot water, add alcohol to the extract, and remove the resulting precipitate. Provided is a neem bark extract having anti-neoplastic activity obtained by collection. The fourth aspect of the present invention is to sequentially pre-treat neem bark with a non-polar organic solvent, a polar organic solvent, and water at room temperature, extract the resulting treated material with hot water, and add alcohol to the extract. The present invention provides a neem bark extract having anti-neoplastic activity obtained by collecting the precipitate produced. Neem's scientific name is Melia azadirachta.
azadirachta) and grows naturally in tropical regions.
It is a woody plant that can reach over 10m. In the present invention, the bark is used as a raw material. The bark is
Dry and shredded products are preferably used. In the present invention, the step of extracting neem bark with hot water involves adding hot water to neem bark, or
It is carried out by adding water to neem bark and heating the mixture to a boil. Heating can be done in a boiling water bath or over an open flame. The extraction time is determined appropriately according to the quality of the raw materials, etc., but is usually 1 to 48 hours.
It's time. After the extraction is completed, the extract mixture is filtered to obtain an extract. In the present invention, a neem bark extract with high pharmacological activity is obtained by adding alcohol to the hot water extract thus obtained and collecting the resulting precipitate.
Alternatively, the hot water extract may be evaporated to dryness, the residue may be dissolved in water again, and alcohol may be added to this aqueous solution to collect the resulting precipitate, or an aqueous alcohol solution may be directly added to the above residue to remove insoluble matter. Neem bark extract can also be obtained by collecting neem bark. Methanol and ethanol are suitable as alcohol, and the alcohol concentration in the extract is 20 to 90.
%, especially around 80%. The precipitate formed in the extract is collected by a conventional method, for example, by centrifugation. The collected precipitate is washed with an aqueous alcohol solution having the same concentration as above, and if desired, washed with nearly 100% alcohol and then ethanol, and dried by freeze drying, ventilation drying or vacuum drying. In the present invention, neem bark is used at room temperature (0 to 40
A neem bark extract with reduced toxicity can be obtained by pre-treating the product with water at a temperature of 30°F (°C), extracting the resulting product with hot water, adding alcohol to the extract, and collecting the resulting precipitate. Furthermore, if neem bark is treated with a polar organic solvent or a non-polar organic solvent prior to the pretreatment with room temperature water, a more highly active neem bark extract is likely to be obtained. Examples of polar organic solvents used in the above pretreatment include methanol, ethanol, propanol,
Alcohols such as n-butanol, pyridine,
Examples include acetone. Examples of non-polar organic solvents include benzene, toluene, xylene,
Examples include n-hexane, chloroform, carbon tetrachloride, and ethyl acetate. Each of the above-mentioned pretreatment steps is carried out by adding a specified solvent to the neem bark as a raw material, and extracting and removing unnecessary components by a conventional method except for water treatment at room temperature or heating. The properties of the neem bark extract in the present invention are as follows. (1) Color and shape Brown powder (2) Infrared absorption spectrum As shown in Figure 1 (Extract of Example 5). IR ν ^KBr _nax cm ^-1 : around 3400, 1620, 1020 (3) Ultraviolet absorption spectrum As shown in FIG. 2 (Extract of Example 5). Water was used as the solvent. UVλ _nax 273nm (4) Sugar content (measured by phenol-sulfuric acid method) 85.3% (converted to soluble starch) (5) Solubility Soluble in water and alkaline aqueous solution, insoluble in methanol, ethanol, benzene, ethyl acetate (6) Acute Toxicity: LD ₅₀ = 400.0 mg/Kg when administered intraperitoneally to ICR male mice. Indicates that the substance is a new substance. The neem bark extract of the present invention is useful for the treatment of various malignant tumors, and its administration forms include parenteral administration by subcutaneous injection, intravenous injection, intramuscular injection, tablets, capsules, granules, etc. Oral administration using powders, syrups, etc. can be mentioned. The extract of the present invention is formulated and administered according to conventional methods. For example, put the dry powder of this extract in a container such as a bias container, prepare physiological saline, glucose solution, or carboxymethylcellulose (CMC) suspension in a separate container such as an ampoule, and suspend and dissolve the powder before use. Inject. Alternatively, it may be injected in the form of an emulsion. For example, in the case of a water-in-oil (W/O) emulsion, a mineral oil such as liquid paraffin, a vegetable oil such as sesame oil or peanut oil, and a surfactant such as sorbitan fatty acid ester are used in combination. Next, the present invention will be explained in more detail with reference to Examples and Formulation Examples. Example 1 200 ml of water was added to 20 g of dried neem bark, and the mixture was heated and boiled over an open flame for about 2 hours to perform hot water extraction. The extraction mixture was filtered to obtain an extract and an extraction residue. 200 ml of water was added to the extraction residue and a hot water extraction operation was performed in the same manner as above. This extraction operation was performed three times in total. The extracts were collected and concentrated to dryness using a rotary evaporator to obtain 1960.5 mg of powder. This powder
1000 mg was dissolved in 200 ml of water, and pure ethanol was gradually added to the resulting aqueous solution at room temperature while stirring. When the ethanol concentration in the aqueous solution reached 80%, the addition was stopped and stirring was continued for a while. The generated precipitate was collected by centrifugation, and the precipitate was diluted with 80% ethanol twice.
After washing twice with pure ethanol and twice with ether, it was vacuum dried to obtain 594.5 mg of neem bark extract as a brown powder. Example 2 Extraction and purification was carried out in the same manner as in Example 1 except that methanol was used instead of ethanol as a precipitant to obtain 520.8 mg of a neem bark extract. Example 3 500 ml of water at 20° C. was added to 50 g of dried neem bark, extraction was carried out at room temperature for about 24 hours, and the resulting extraction mixture was filtered. Add 500 ml of water at 20℃ to the extraction residue.
ml was added and the extraction process was performed in the same manner as above. This extraction process operation was performed three times in total. 500 ml of water was added to the obtained extraction residue, and hot water extraction was performed while boiling on a gas burner for about 2 hours. The extraction mixture was filtered to obtain an extract and an extraction residue. This hot water extraction operation was performed three times in total. The extract was collected and concentrated to dryness using a rotary evaporator to give 1418.0 mg.
powder was obtained. 1000 mg of the obtained powder was dissolved in 200 ml of water, and pure ethanol was gradually added to the obtained aqueous solution at room temperature while stirring. When the ethanol concentration in the aqueous solution reached 80%, the addition was stopped and stirring was continued for a while. The resulting precipitate was treated in the same manner as in Example 1 to obtain 671.3 mg of neem bark extract. Example 4 500 ml of methanol was added to 50 g of dried neem bark and the extraction mixture was filtered for about 24 hours at room temperature. 500 ml of methanol was added to the extraction residue, and the extraction process was performed in the same manner as above. This extraction process operation was performed three times in total. The obtained extraction residue was treated in the same manner as in Example 3. That is, the extraction residue is 20
The extract was extracted with water at ℃, the extraction residue was extracted with hot water, and the extracts were collected and concentrated to dryness to obtain 1409.0 mg of powder. 1000 mg of this powder was dissolved in 200 ml of water, pure ethanol was added to the resulting aqueous solution, and the resulting precipitate was collected to obtain 668.2 mg of neem bark extract. Example 5 In Example 4, instead of concentrating the hot water extract to dryness, the extract was concentrated to 300 ml, and pure ethanol was added to this concentrated liquid. 1341.1 mg of extract was obtained. Example 6 The same procedure as in Example 4 was performed except that ethanol was used instead of methanol as the extraction treatment agent to obtain 1371.8 mg of hot water extract powder.
1000 mg was dissolved in 200 ml of water, pure ethanol was added to the resulting aqueous solution, and the resulting precipitate was collected to obtain 654.8 mg of neem bark extract. Example 7 500 ml of benzene was added to 50 g of dried neem bark, extraction was carried out at room temperature for about 24 hours, and the extracted mixture was filtered. The obtained extraction residue was treated in the same manner as in Example 4. That is, the extraction residue was extracted with methanol, the residue was extracted with water at 20°C, the extraction residue was extracted with hot water, and the extract was concentrated to dryness.
1353.3 mg of powder was obtained. 1000mg of this powder in 200ml of water
ml, add pure ethanol to the resulting aqueous solution, collect the resulting precipitate, and extract the neem bark extract.
695.3 mg was obtained. Example 8 Instead of adding pure ethanol to the hot water extract aqueous solution so that the ethanol concentration was 80%, the same concentration was added.
Neem bark extract
249.0 mg was obtained. Example 9 Instead of adding pure ethanol to the hot water extract aqueous solution so that the ethanol concentration was 80%, the same concentration was added.
Other than adding pure ethanol to make it 50%,
Neem bark extract was prepared in the same manner as in Example 4.
272.2mg was obtained. Example 10 Instead of adding pure ethanol to the hot water extract aqueous solution so that the ethanol concentration was 80%, the same concentration was added.
Neem bark extract was prepared in the same manner as in Example 1 except that pure ethanol was added to make the concentration 25%
227.0mg was obtained. Example 11 The same operation as in Example 4 was performed except that instead of adding pure ethanol to the hot water extract aqueous solution so that the ethanol concentration was 80%, pure ethanol was added so that the same concentration was 25%. neem bark extract
243.8mg was obtained. Formulation Example 1 200 mg of the neem bark extract obtained in Example 1 was dissolved in 100 ml of a sterile 5% glucose solution for injection, and 1 ml of this solution was aseptically dispensed into vials and freeze-dried. In this way, 2 mg in 1 vial
A formulation containing neem bark extract was obtained. Before use, dissolve in distilled water for injection. Formulation Example 2 A vial formulation was prepared in the same manner as in Formulation Example 1 above. However, sterile 5% glucose solution for injection
100 ml of physiological saline was used instead of 100 ml. Before use, dissolve in distilled water for injection. Specific Effects of the Invention The anti-neoplastic effects of the neem bark extracts obtained in each of the above Examples were measured. Test example 1 Effect on Sarcoma 180 ascites cancer (sample preparation) Phosphate buffered saline (manufactured by Gibco, phosphoric acid 9.5 m
Each fraction sample was dissolved in a solution of 0.5% carboxymethylcellulose (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 along with ascites and diluted appropriately with physiological saline to adjust the cell number to 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 into the abdominal cavity once a day for 4 consecutive days using a syringe. 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 ascites fluid was sucked into a hematocrit tube and centrifuged at low temperature using a hematocrit measuring rotor to obtain an acytocrit value (percentage of cancer cells in the ascites), which corresponds to the hematocrit value of blood.
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. In the table, the samples are indicated by example numbers, which indicates that the neem bark extract obtained in the corresponding example was used as the sample. 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) 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 calculating the ratio (T/C) between this weight and that of the control. The control cancer tissue weight is
It was 1.5 to 3.5 g. 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 extremely effective (〓). The results are shown in Table 1.

【table】

【table】

[Table] Furthermore, the minimum effective concentration of the neem bark extract of the present invention against Sarcoma 180 was as follows.

[Table] From the above results, it is clear that the neem bark extract of the present invention has strong activity against Sarcoma 180 tumor. In particular, the extract of the present invention
Compared to the hot water extract concentrated to dryness, the activity against ascites cancer is weaker, but the activity against solid cancer is significantly stronger.

[Brief explanation of the drawing]

FIG. 1 shows the infrared absorption spectrum of the neem bark extract obtained in Example 5, and FIG. 2 shows the ultraviolet absorption spectrum of the same substance.

Claims (1)

[Claims] 1. Anti-malignant properties obtained by extracting the bark of neem (Melia azadirachta, hereinafter the same) with hot water, adding alcohol to the resulting extract, and collecting the resulting precipitate. Neem bark extract with neoplastic properties. 2. The neem bark extract having anti-malignant neoplastic activity according to claim 1, wherein the alcohol is ethanol. 3. The neem bark extract having anti-neoplastic activity according to claim 1, wherein the alcohol is methanol. 4 Anti-malignant neoplasm obtained by pre-treating neem bark with water at room temperature, extracting the resulting treated material with hot water, adding alcohol to the resulting extract, and collecting the resulting precipitate. Neem bark extract with action. 5 Neem bark is sequentially pretreated with a polar organic solvent and water at room temperature, the resulting treated material is extracted with hot water, alcohol is added to the resulting extract, and the resulting precipitate is collected. Neem bark extract with anti-neoplastic activity. 6. The neem bark extract having anti-neoplastic activity according to claim 5, wherein the polar organic solvent is alcohol. 7. The neem bark extract having anti-neoplastic activity according to claim 6, wherein the alcohol as the polar organic solvent is methanol. 8. The neem bark extract having anti-neoplastic activity according to claim 6, wherein the alcohol as the polar organic solvent is ethanol. 9 Neem bark is sequentially pretreated with a non-polar organic solvent, a polar organic solvent, and water at room temperature, the obtained treated material is extracted with hot water, alcohol is added to the obtained extract, and the resulting precipitate is extracted. A neem bark extract with anti-neoplastic activity obtained by collecting neem bark. 10. The neem bark extract having anti-neoplastic activity according to claim 9, wherein the non-polar organic solvent is benzene.