JPS6153333B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION TECHNICAL FIELD The present invention relates to a novel extract of Tuluna. The Tuluna extract of the present invention has anti-neoplastic activity and is useful as an anti-tumor agent. Prior Art Tsuruna is a Japanese folk medicine, also known as Hamadishiya, and is said to be good for stomach cancer and stomach ulcers. but,
Its ingredients and pharmacological effects are unknown, and a book on medicinal plants published in recent years states that it is said to be ``effective against stomach cancer, but it is doubtful.'' Published April 1, 1955, pages 21-22]. Purpose of the Invention As a result of intensive research into the pharmacological effects of the components contained in trunna, the present inventors have discovered that the ingredients obtained by extracting the whole trunna plant using a specific method have excellent anti-neoplastic effects. I learned that I have it. Accordingly, it is an object of the present invention to provide a novel Tulna extract that is effective in the treatment of malignant neoplasms, particularly malignant tumors. DETAILED DESCRIPTION OF THE INVENTION The tulna extract provided by the present invention includes the following. (1) A trunna extract with anti-neoplastic activity obtained by extracting the whole trunna plant with hot water and purifying the resulting extract by alcohol precipitation or dialysis membrane method. (2) The whole plant of Trunna is pretreated with water at room temperature, the resulting treated material is extracted with hot water, and the resulting extract is purified by alcohol precipitation method or dialysis membrane method. Tuluna extract with malignant neoplastic effects. (3) Pre-treat the whole plant of Trunna sequentially with a polar organic solvent and water at room temperature, extract the resulting treated material with hot water, and purify the resulting extract using alcohol precipitation or dialysis membrane method. A trunna extract with anti-neoplastic activity obtained by (4) Pre-treat the whole plant of Trunna sequentially with a non-polar organic solvent, a polar organic solvent, and water at room temperature, extract the resulting treated material with hot water, and use the alcohol precipitation method or A trunna extract with anti-neoplastic activity obtained by purification using the dialysis membrane method. Trunna is a member of the Trunaceae family (Garnetaceae,
Aizoaceae), the scientific name is Tetragonia
tetragonoides (PALL.) O.Kuntze. In the present invention, the whole plant is used as an extraction raw material. In the present invention, the step of extracting the whole trunna plant with hot water is carried out by adding hot water to the trunna whole plant, or by adding water to the trunna whole plant and heating and boiling the mixture. . Heating can be carried out in a boiling water bath or over an open fire. 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 process is completed, the extraction mixture is filtered to obtain an extract. The thus obtained extract is subjected to alcohol precipitation treatment or dialysis membrane treatment to obtain the desired extract. That is, when performing alcohol precipitation treatment, alcohol such as methanol, ethanol, etc. is added to the above-mentioned extract, and the resulting precipitate is collected. Alcohol has an alcohol concentration of 50 to 90% in the extract, especially
It is desirable to add an amount that makes it around 80%. The precipitate formed in the extract is collected by a conventional method, for example, by centrifugation, and dried by freeze drying, ventilation drying or vacuum drying. When performing dialysis membrane treatment, the extract is treated with a dialysis membrane and the active ingredients are collected from the dialyzed fluid. Dialysis membranes include Visking tube (Union Carbide Corp.) or Spectra Por (Spectrum Medical Industries).
Medical Industries Co.) product is used.
Dialysis is performed against water, and the desired extract can be obtained by concentrating the dialyzed solution to dryness or freeze-drying it. In the above-mentioned alcohol precipitation treatment or dialysis membrane treatment, a dry extract is obtained by evaporating to dryness, freeze-drying or spray-drying the extract of the whole plant of Tulna, and the above-mentioned purification treatment is performed on the resultant which is dissolved in water again. It's okay. Furthermore, in the present invention, by pre-treating the whole Trunna plant with water at room temperature (0 to 40° C.) and extracting the obtained treated material with hot water, an extract with fewer impurities can be obtained. Further, if the whole plant, preferably dried, is treated with a polar organic solvent prior to the pretreatment with room temperature water, an extract of higher purity can be obtained. Examples of polar organic solvents include alcohols such as methanol, ethanol, propanol, n-butanol, pyridine, acetone, and the like. In the present invention, further, prior to the pretreatment with the polar organic solvent, the whole plant of Trunna is treated with a non-polar organic solvent such as benzene, toluene, xylene, n-hexane, chloroform, carbon tetrachloride, ethyl acetate, etc. It can also be pretreated. Each of the above pretreatment steps is carried out by adding an appropriate amount of solvent to the raw material and extracting and removing unnecessary components at room temperature according to a conventional method. Extracts using polar or non-polar organic solvents described above did not show any anti-neoplastic activity, but extracts using room temperature water showed an inhibitory effect on ascites cancer. The characteristics of the Tulna extract in the present invention are as follows. (1) Color and shape Pale yellowish brown powder (2) Ultraviolet absorption spectrum As shown in Figure 1 (extract of Example 4) (3) Infrared absorption spectrum As shown in Figure 2 (Example 4) (4) Molecular weight From the results of dialysis using a Visking tube, it ranges from several thousand to more than 10,000 (5) Sugar content (measured by the phenol-sulfuric acid method) 51.8% (converted to soluble starch) (6) Solubility Possible in water Soluble, insoluble in ethanol, benzene, and ethyl acetate (7) Acute toxicity As a result of intraperitoneal administration to ICR male 4-week-old mice,
_LD50 was 450mg/Kg. The Tulna extract of the present invention is useful for the treatment of various malignant tumors, and its administration form includes parenteral administration such as subcutaneous injection, intravenous injection, and intramuscular injection, or tablets, capsules, granules, and powders. Oral administration using syrup, etc. can be mentioned. The dosage of this extract depends on the route of administration, the age of the patient,
1 for adult males, depending on weight and symptoms
Approximately 1 to 5 g per day. 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 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 A hot water extraction treatment was carried out by adding 20 kg of dried whole plant of Tulna to 20 kg of water and heating the mixture in a boiling water bath for about 5 hours. The extraction mixture was filtered, 20 g of water was added to the extraction residue, and hot water extraction was performed in the same manner as above. This extraction operation was performed three times in total, and the extracts were collected and concentrated to dryness using a rotary evaporator to obtain 88.5 g of a hot water extract as a powder. 10.0 g of the hot water extract thus obtained was dissolved in 500 ml of water, and 99% ethanol was slowly added to this aqueous solution. When the ethanol concentration in the aqueous solution reached 80%, the addition was stopped and the mixture was stirred for a while. The generated precipitate was collected by centrifugation and diluted with 80% ethanol.
The extract was washed twice with 99% ethanol and twice with ether, and dried under vacuum to obtain 7.22 g of Tuluna extract. Example 2 9.5 g of a hot water extract of the whole plant of Tuluna obtained in the same manner as in Example 1 was dissolved in 500 ml of water, and the resulting aqueous solution was placed in a Visking tube and dialyzed against water. Concentrate the dialysis fluid to dryness and extract Trunna extract.
4.21g was obtained. Example 3 Add 2 kg of room temperature water to 2 kg of dried whole plant of trunna,
Pre-extraction treatment was carried out for about 2 days, and the resulting extraction mixture was filtered. Water at room temperature (20 g) was added to the extraction residue and pre-extraction treatment was carried out in the same manner as above. This extraction pretreatment operation using room temperature water was performed three times in total. 20 g of water was added to the pre-extraction treatment residue, and hot water extraction was performed in a boiling water bath for about 5 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 86.9 g of hot water extract. Add 10.0g of this hot water extract to 500ml of water.
ml, and the resulting aqueous solution was subjected to alcohol precipitation in the same manner as in Example 1 to obtain 6.73 g of Tuluna extract. Example 4 20 g of methanol was added to 2 kg of dried Tulna whole plant, pre-extraction treatment was carried out at room temperature for about 24 hours, and the extraction mixture was filtered. Methanol 20 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. The obtained extraction residue was pre-extracted with water at room temperature in the same manner as in Example 3. Indicated). The extraction residue was extracted with hot water in the same manner as in Example 3 to obtain 80.1 g of hot water extract.
10.0 g of this hot water extract was dissolved in 500 ml of water, and the resulting aqueous solution was treated with 99% ethanol in the same manner as in Example 1 to obtain 6.51 g of Thurna extract. Example 5 Hot water extract of the whole Trunna plant obtained in Example 4
Dissolve 10.0g in 500ml of water, slowly add 99% ethanol to this aqueous solution, stop adding ethanol when the ethanol concentration of the aqueous solution reaches 50%, collect the formed precipitate by centrifugation, and extract 4.07 I got g. Example 6 Hot water extract of the whole Trunna plant obtained in Example 4
500 ml of 80% ethanol was added to 10.0 g, thoroughly suspended, and the purification operation was performed while stirring. The insoluble material was removed, 50 ml of 80% ethanol was added thereto, and the same purification procedure was performed to obtain 8.52 g of Tuluna extract. Example 7 Hot water extract of the whole Trunna plant obtained in Example 4
9.5 g was dissolved in 500 ml of water, and the resulting aqueous solution was placed in a Visking tube and dialyzed against water. The dialyzed fluid was concentrated to dryness to obtain 5.74 g of Tuluna extract. Example 8 The same procedure as in Example 4 was carried out except that the pre-extraction treatment was performed with ethanol instead of methanol, to obtain 82.5 g of Tuluna extract. Example 9 Add 20 kg of benzene to 2 kg of dried trunna whole plant,
Extraction pretreatment was carried out at room temperature for about 24 hours, and the extraction mixture was filtered. Benzene 20 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. The obtained extraction residue was pretreated with methanol and then water at room temperature in the same manner as in Example 4, the extraction residue was extracted with hot water, and the hot water extract was treated in the same manner as in Example 4 to obtain 80.3 g of Tuluna extract. Obtained. Example 10 The same procedure as in Example 9 was carried out except that the pre-extraction treatment was performed with ethanol instead of methanol, to obtain 81.9 g of Tuluna extract. Formulation Example 1 1000 mg of Tuluna extract 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. In this way a formulation containing 10 mg of tulna extract in one vial was 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 effects of the Tulna extract obtained in the above examples were measured. Test Example 1 Effect on L-5178Y cells (cell preparation) Mouse L-5178Y cells cultured for 4 days in RPMI-1640 medium containing 10% fetal bovine serum were 1.0×10 ⁵
50 µm per well (250 µm volume) was prepared in a 96-well U-bottom microplate. (Efficacy evaluation method) Pour 50μ of the sample dissolved in the medium into the above microplate per hole, and place it in a carbon dioxide incubator at 37℃.
Cultured for 48 hours. After culturing, count the number of cells in each well.
The number was compared with that of a control without a sample, and the concentration required to kill 50% of the cells was calculated and defined as ID ₅₀ . The results are shown in Table 1. In the table, the samples are indicated by Example numbers, which indicates that the Tuluna extract obtained in the corresponding Example was used as the sample. The same applies to Table 2.

[Table] Test example 2 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% 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 2 shows the results.
Shown below. Test Example 3 Effect on Sarcoma 180 cancer cells (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 2. 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 2.

[Table] From Tables 1 and 2 above, it is clear that the Tuluna extract of the present invention has extremely low cytotoxicity to established mouse lymphocytes L-5178Y and exhibits strong activity against ascites cancer and solid cancer. be. In addition, a room temperature water extract of the whole plant Trunna shows activity against ascites cancer, but has low cytotoxicity against L-5178Y and low activity against solid cancer. Example 4
Minimum effective concentration of Tulna hot water extract (mouse)
The amount was 0.99 mg/Kg for ascites cancer and 0.98 mg/Kg for solid cancer.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum of the Tuluna extract obtained in Example 4, and FIG. 2 shows the infrared absorption spectrum of the same substance.

Claims (1)

[Scope of Claims] 1. An extract having an anti-neoplastic effect obtained by extracting the whole plant of Tuluna with hot water and purifying the obtained extract by alcohol precipitation or dialysis membrane method. 2. The extract having anti-neoplastic activity according to claim 1, wherein the alcohol precipitation method is a precipitation method using ethanol. 3. The Tulna extract having anti-neoplastic activity according to claim 2, which is a precipitation method in which ethanol is added to the extract in an amount to give a concentration of 80%. 4. The extract having anti-neoplastic activity according to claim 1, wherein the dialysis membrane method is a method using a Visking tube. 5 Anti-malignant new product obtained by pre-treating the whole plant of Trunna with water at room temperature, extracting the obtained treated material with hot water, and purifying the obtained extract by alcohol precipitation method or dialysis membrane method. Tulna extract with biological effects. 6. The whole plant of Trunna is sequentially pretreated with a polar organic solvent and water at room temperature, the obtained processed material is extracted with hot water, and the obtained extract is purified by alcohol precipitation method or dialysis membrane method. The resulting trunna extract with anti-neoplastic activity. 7. The extract having anti-neoplastic activity according to claim 6, wherein the polar organic solvent is alcohol. 8. The trunna extract having anti-malignant neoplastic activity according to claim 7, wherein the alcohol is methanol. 9. The extract having anti-malignant neoplastic activity according to claim 7, wherein the alcohol is ethanol. 10 Pre-treat the whole plant of Trunna sequentially with a non-polar organic solvent, a polar organic solvent, and water at room temperature, extract the obtained treated material with hot water, and use the alcohol precipitation method or dialysis membrane to extract the obtained extract. A trunna extract with anti-neoplastic activity obtained by purification using a method. 11. The extract having anti-neoplastic activity according to claim 10, wherein the non-polar organic solvent is benzene.