JPS6153335B2 - - Google Patents

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel hot water extract of melium bark and its purified product. The extract of the present invention is useful as an anti-neoplastic agent, especially an anti-tumor agent. Prior Art Melium bark is called kurenpi in Chinese medicine, and is decocted and used to exterminate roundworms, hookworms, and tapeworms, and to treat malarial fever. However, it has not been previously known that the bark of maize contains components that have anti-neoplastic effects. Purpose of the Invention As a result of extensive research into the pharmacological effects of the components contained in the bark of Chilium, the present inventors have found that a hot water extract obtained by extracting the bark of Chilium using a specific method is an excellent anti-malignant new product. I learned that it has biological effects. Therefore, it is an object of the present invention to provide a hot water extract of melium bark having anti-neoplastic activity. That is, the present invention provides a hot water extract of melium bark as described in each of the following items. (1) A hot water extract of chili bark obtained by pre-extracting chili bark with water or a polar organic solvent at room temperature and extracting the resulting extraction residue with hot water. (2) The melium bark hot water extract according to item 1, wherein the polar organic solvent is alcohol. (3) The melium bark hot water extract according to item 2, wherein the alcohol is methanol. (4) Pre-extraction treatment of melium bark with water or polar organic solvent at room temperature, extraction treatment of the obtained extraction residue with hot water, and purification of the obtained extract by alcohol precipitation method or dialysis membrane method. Purified hot water extract of melium bark. (5) The purified meli bark hot water extract according to item 4, wherein the alcohol precipitation method is a precipitation method using ethanol. (6) The purified hot water extract of melid bark according to item 5, which is a precipitation method in which ethanol is added to the extract in an amount to give a concentration of 50 to 95%. (7) The purified meli bark hot water extract according to item 4, wherein the dialysis method uses a dialysis membrane with a molecular weight cutoff of 6,000 to 10,000. (8) The permeable membrane is Spectrapore 6 (Spectra
Por6) (a product of Spectrum Medical Industries). The hot water extract of maize bark obtained according to the present invention is a new substance and is different from the conventionally known meli bark extract, that is, a substance obtained by directly extracting the meli bark with hot water. Detailed Description of the Invention The hot water extract of chili bark of the present invention is obtained by first pre-extracting chili bark with water or a polar organic solvent at room temperature, extracting the obtained extraction residue with hot water, and extracting it from the extract. Obtained by removing water. In the present invention, the term "meli bark" refers to melium (Melia azedarach L. var.
Makino) bark. The bark is desirably dried and shredded, and commercially available bark is suitably used. The extract of the present invention is added to the above bark at room temperature (0-40
℃) of water or a polar organic solvent and perform an extraction pretreatment at room temperature for several hours to 2 days, or sequentially perform an extraction pretreatment with a polar organic solvent and water at room temperature, and add hot water to the resulting extraction residue; Alternatively, it can be obtained by adding water to the above extraction residue, heating the mixture to boiling for extraction treatment, and removing water from the liquid after filtration. Examples of polar organic solvents used for extraction pretreatment in the present invention include methanol, ethanol,
Examples include alcohols such as n-propanol, isopropanol, and n-butanol, pyridine, and acetone. The step of adding water to the extraction residue and heating it can be performed in a boiling water bath or over an open flame. The extraction treatment time using hot water is determined as appropriate depending on the type and quality of the raw materials, but it is usually several hours to two hours.
About a day. After the extraction process is completed, the extraction mixture is filtered to obtain an extract. The desired extract is obtained by drying the extract thus obtained by concentration drying, freeze drying, spray drying, or the like. In the present invention, a purified extract can also be obtained by subjecting the hot water extract to alcohol precipitation treatment or dialysis membrane treatment. When alcohol precipitation is performed, alcohol such as methanol, ethanol, etc. is added to the above-mentioned extract, and the resulting precipitate is collected. The alcohol concentration in the extract is 50-95%, especially 80%.
It is desirable to add amounts that will be around the same amount. The precipitate formed in the extract is collected by a conventional method, for example, by filtration or centrifugation, and dried by freeze drying, ventilation drying, vacuum drying, or the like. When performing dialysis membrane treatment, the above-mentioned extract is placed in a dialysis membrane, dialyzed against water, and the dialyzed solution is dried by concentration to dryness, freeze-drying, etc. to obtain the desired extracted and purified product. The dialysis membrane has a molecular weight cutoff of 6,000 to 10,000, such as Spectra Por 1 or 6 (Spectrum Medical Industries Co.).
) product] or Visking tube (Visking
tube) [Union Carbide
Corp.) products] are used. In the above alcohol precipitation treatment or dialysis membrane treatment, the hot water extract of melium bark is evaporated to dryness,
An extract may be obtained by freeze-drying or spray-drying, and the above-mentioned purification treatment may be performed by redissolving the extract in water. Furthermore, in the present invention, prior to the extraction pretreatment with water or a polar organic solvent at room temperature, a non-polar organic solvent such as benzene, toluene, etc.
Extraction pretreatment can also be performed with xylene, n-hexane, chloroform, carbon tetrachloride, ethyl acetate, or the like. The properties of the hot water extract of melium bark in the present invention are as follows. (1) Color and shape Light brown powder (2) Ultraviolet absorption spectrum As shown in Figure 1 (Extract of Example 8). UVλ ^KBr _nax 276nm (3) Infrared absorption spectrum As shown in Figure 2 (Extract of Example 8). IRνmaxcm ^-1 : around 3400, 2920, 1610 (4) Solubility Soluble in water, insoluble in benzene, ethanol, and ethyl acetate. (5) Molecular weight More than 10,000 based on the results of Spectrapore dialysis (6) Acute toxicity LD ₅₀ 800mg/Kg Intraperitoneal administration to male ICR mice weighing 20±1gr For reference, a publicly known product obtained by directly extracting the bark of Melium with hot water The ultraviolet absorption spectrum and infrared absorption spectrum of the extract are shown in FIGS. 3 and 4, respectively. A comparison between FIG. 1 and FIG. 3 and between FIG. 2 and FIG. 4 shows that the extract of the present invention is a different substance from known extracts. The melium bark extract of the present invention has an inhibitory effect on various malignant neoplasms. For example, as described below, it has a suppressive effect on Sarcoma 180 ascites cancer and solid cancer, P388 murine leukemia, and the like. Therefore, it is useful for treating various malignant tumors. When administering the extract of the present invention, for example, parenteral administration via subcutaneous injection, intravenous injection, intramuscular injection, or oral administration via tablets, capsules, granules, powders, syrups, etc. can be mentioned. . The dosage of this extract varies depending on the route of administration, age, weight, and symptoms of the patient, but is approximately 1 to 5 g per day for an adult male. 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 vial, prepare physiological saline, glucose solution, or carboxymethyl cellulose (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 Add 300 ml of methanol to 30.0 g of commercially available turmeric.
Extraction pretreatment was performed for 24 hours and the extraction mixture was filtered. 300 ml of methanol was added to the extraction residue and pre-extraction treatment was performed in the same manner as above. This extraction pretreatment operation was performed three times in total. Water 300% to the obtained extraction residue
ml and hot water extraction was carried out in a boiling water bath for about 2 hours. This hot water extraction operation was performed three times in total, and the resulting hot water extracts were collected and concentrated to dryness using a rotary evaporator to obtain 2371.7 mg of hot water extract. Example 2 The extraction procedure was carried out in the same manner as in Example 1, except that water at room temperature was used instead of methanol for extraction pretreatment to obtain 602.4 mg of a hot water extract. Example 3 300 ml of methanol was added to 30.0 g of commercially available Chimundan bark, pre-extraction treatment was carried out at room temperature for about 24 hours, and the extraction mixture was filtered. 300 ml of methanol was added to the extraction residue, and extraction pretreatment was performed in the same manner as above. This extraction pretreatment operation was performed three times in total. The obtained extraction residue was pretreated with room temperature water in the same manner as in Example 2, and the obtained extraction residue was extracted with hot water to obtain a hot water extract.
577.8mg was obtained. Example 4 Extraction pretreatment and extraction treatment operations were carried out in the same manner as in Example 3, except that dried melium bark was used instead of commercially available bittern bark, and hot water extract 309.1 was obtained.
I got mg. Example 5 300 ml of benzene was added to 30.0 g of commercially available kumungha,
Extraction pretreatment was performed for 8 hours at room temperature. The extraction mixture was filtered, 300 ml of benzene was again added to the residue, and extraction pretreatment was performed in the same manner as above. This operation was performed three times in total. The thus obtained extraction residue was subjected to extraction pretreatment using methanol and then room temperature water in the same manner as in Example 3, and the obtained extraction residue was extracted with hot water to obtain 598.4 mg of a hot water extract. Example 6 Extraction pretreatment and extraction treatment were carried out in the same manner as in Example 5, except that dried melium bark was used instead of commercially available chili bark, to obtain 313.2 mg of a hot water extract. Example 7 1.00 g of hot water extract of chili bark obtained by the same procedure as in Example 1 was dissolved in 50 ml of water, and 99% ethanol was slowly added to this aqueous solution until the ethanol concentration in the aqueous solution was 50%. When the temperature reached 100, the addition was stopped and the mixture was stirred for a while. The generated precipitate was collected by centrifugation, washed successively with 50% ethanol, 99% ethanol and ether, and dried under vacuum to obtain 277.1 mg of a purified hot water extract of melium bark. Example 8 Example 7 except that in Example 7, the ethanol concentration in the aqueous solution of melium bark extract was 80%.
In exactly the same manner as above, 425.3 mg of purified meli bark hot water extract was obtained. Example 9 1.00 g of hot water extract of chili bark obtained in the same manner as in Example 1 was made into a fine powder, 80% ethanol was added thereto, and the mixture was thoroughly stirred and suspended. Collect the insoluble portion by centrifugation, 80% ethanol, 99%
% ethanol and ether, and dried under vacuum to obtain 626.4 mg of purified meli bark hot water extract. Example 10 1.00 g of the hot water extract of chili bark obtained by the same procedure as in Example 1 was dissolved in 200 ml of water, and the resulting aqueous solution was mixed with Spectrapore 6 (molecular weight cutoff).
10,000) and dialyzed against water. Concentrate and dry the dialysis fluid to obtain a purified hot water extract from chili bark.
298.7mg was obtained. Formulation Example 1 Meli bark hot water extract obtained in Example 1
1000 mg was dissolved in 500 ml of a sterile 5% glucose solution for injection, and 5 ml of this solution was aseptically dispensed into vials and freeze-dried. A formulation containing 10 mg of melium bark extract in one vial was thus obtained.
Before use, dissolve in distilled water for injection. Formulation Example 2 A vial preparation was prepared in the same manner as in Formulation Example 1 above. However, sterile 5% glucose solution for injection 500
500 ml of physiological saline was used instead of ml. When using,
Use by dissolving in distilled water for injection. Specific Effects of the Invention The anti-neoplastic effect of the hot water extract of chili bark obtained in the above example was measured. Test example 1 Effect on Sarcoma 180 ascites cancer (sample preparation) Phosphate buffered saline (manufactured by Gibco, phosphoric acid 9.5m
Each fraction 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 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. 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 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 determining 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. As shown in Table 1, the extract of the present invention
Among the 180 transplanted cancers, it has a particularly strong suppressive effect on solid tumors.

【table】

[Table] The minimum effective dosage of the hot water extract of melium bark (extract purified product of Example 8) is as follows. Sarcoma 180 Ascites Cancer 50mg/Kg Sarcoma 180 Solid Cancer 22mg/Kg Test Example 3 Effect on P388 Mouse Leukemia Inject 0.1ml of P388 cell suspension into the peritoneal cavity of a 4-5 week old male CDF ₁ mouse (number of cells: 1 x ¹⁰⁶ ) ) was inoculated. twenty four
After that time, 0.1 ml of a 0.5% carboxymethyl cellulose sodium salt suspension containing a predetermined amount of the sample was intraperitoneally administered once a day for a total of 5 days. The average survival days were determined, and the ratio (T/C%) between the treated group (T) and the control group (C) was determined. The average survival time of the control group was 7-12 days. It can be said that there is activity when T/C≧125%. The results are shown in Table 2.

[Table] Test Example 4 Effect on established solid cancers 1 x 10 ⁷ Sarcoma 180 cells were injected into ICR mice (5
The animals were transplanted subcutaneously to the back and reared as they were for 2 weeks. Sarcoma 180 implanted subcutaneously is 1000-1500
After confirming that the plant has grown to a size of ³ mm (approximately 12 x 15 mm), from the 14th day after transplantation, the purified meli bark hot water extract obtained in Example 8 was intraperitoneally administered once a day.
150mg/Kg was administered for 5 days. Short axis of tumor part (a
mm) and major axis (bmm) with a caliper, 1/2a ² b
(mm ³ ) was defined as the tumor volume. The results are shown in Figure 5. Furthermore, on the 28th day after transplantation, the tumor area was cut out and its weight was measured. The tumor area of untreated mice was 7.39 g, while the tumor area of treated mice was 0.25 g (T/C = 5.2
%). Furthermore, the tumors in 3 out of 5 mice disappeared. From the graph of FIG. 5 and the above results, it is clear that the hot water extract of melium bark of the present invention has a remarkable effect on colonized solid cancer.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum of the purified meli bark extract obtained in Example 8, and FIG. 2 shows the infrared absorption spectrum of the same substance. Figure 3 shows the ultraviolet absorption spectrum of a known extract obtained by directly extracting the bark of melium with hot water, and Figure 4 shows the infrared absorption spectrum of the same substance. FIG. 5 is a graph showing the results of Test Example 4.

Claims (1)

[Scope of Claims] 1. A hot water extract of chili bark obtained by pre-extracting chili bark with water or a polar organic solvent at room temperature and extracting the resulting extraction residue with hot water. 2. The melium bark hot water extract according to claim 1, wherein the polar organic solvent is alcohol. 3. The melium bark hot water extract according to claim 2, wherein the alcohol is methanol. 4. Neil obtained by pre-extracting melium bark with water or a polar organic solvent at room temperature, extracting the resulting extraction residue with hot water, and purifying the resulting extract by alcohol precipitation or dialysis membrane method. Purified hot water extract of bark. 5. The purified hot water extract of melium bark according to claim 4, wherein the alcohol precipitation method is a precipitation method using ethanol. 6. Claim 5, which is a precipitation method in which ethanol is added to the extract in an amount to give a concentration of 50 to 95%.
Purified hot water extract of melium bark as described in . 7. The purified melium bark hot water extract according to claim 4, wherein the dialysis method is a method using a dialysis membrane with a molecular weight cutoff of 6,000 to 10,000. 8 The dialysis membrane is Spectrapore 6 (Spectra
Por6) (a product of Spectrum Medical Industries).