JPS5935891B2 - Anti-neoplastic agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to neem (Melia azadirachta).
The present invention relates to an anti-neoplastic agent containing as an active ingredient a substance obtained by extracting the bark of Melia azadirakucu with an appropriate solvent.

Anti-neoplastic agents (anticancer agents) are generally classified into alkylating agents, antimetabolites, antibiotics, and natural products (plant ingredients, etc.) in terms of their mechanism of action or material origin, and many formulations for each have been developed. There is.

The present invention relates to an anti-neoplastic agent containing an extract from a natural product (plant component) as an active ingredient.

Generally, in natural product chemistry, substances are extracted using solvents with low polarity such as benzene, ether, and ethyl acetate, and the chemical structures of many of the trough components of substances have been determined.

Recently, many host-mediated anticancer drugs or immunostimulants have been developed by extracting natural products with relatively highly polar solvents such as water or hot water, without using conventional low-polar solvents. is taken.

Examples of these related to the present invention include (A) Japanese Patent Publication No. 52
-28853, (B) Special Publication No. 52-28854, (
C) Special Publication No. 53-10124, (D) Special Publication No. 1983
-10125, (Keyaki Mochiko Sho 53-13689, etc.) are known.

In (A) ~, the medicinal ingredients are extracted from the bark, leaves, flowers, fruits, branches, roots, and resin of neem using a hydrophilic solvent such as methyl alcohol or ethyl alcohol, water, hot water, etc. , (E), the medicinal ingredients are extracted with a hydrophobic solvent such as benzene or ethyl alcohol.

Regarding its medicinal efficacy, (3) for acne, (I3) for rough skin, (C) and ) for Staphylococcus aureus 209P, (E) for Staphylococcus aureus 209 Pl, Bacillus subtilis and Purcella. - Although it has been reported that Avolux has medicinal efficacy, there have been no reports yet that it has activity against malignant neoplasms as in the present invention.

In addition, among the substances that are generally extracted with relatively highly polar solvents, there are many substances that are extracted with less polar solvents. 4 If you suddenly extract with a highly polar solvent as in the above known example, In addition to the extracted components, many components are extracted at the same time, and it is not only unclear which components among the extracted components exhibit effective medicinal effects, but also the content of medicinal components tends to be low, thus impractical. It becomes fragile.

Neem is a perennial plant that grows in tropical regions and reaches a height of more than 100 ft., and the present inventors have confirmed that neem extract, which has anti-cancer properties as will be detailed later, is particularly noticeable in its bark. .

Furthermore, it was confirmed that the target active ingredient could be extracted at a higher concentration by extracting the bark in order of decreasing dielectric constant (polarity).

The substance from which the target component was extracted at a higher ratio in this way was obtained from Mouse L-51 as described in detail later.
Efficacy tests were conducted on 78Y cells, mouse Sarcoma 180 ascites cancer, and mouse Sarcoma 180 solid cancer, and it was confirmed that the drug exhibited effective effects.

As a result of conducting the same tests as the anti-neoplastic agent of the present invention on other existing effective anti-cancer drugs,
It was confirmed that this drug has medicinal efficacy comparable to those of these drugs, leading to the present invention.

An object of the present invention is to perform an appropriate extraction treatment on neem bark to extract the target active ingredient at the highest possible concentration, and to provide an anti-neoplastic agent using this extract as a medicinal ingredient.

The present invention extracts neem bark with water at 0 to 40°C,
To provide an anti-neoplastic agent containing as an active ingredient a substance obtained by extracting the extraction residue with hot water at 40 to 100°C.

In addition, before treating neem bark with water at 0 to 40°C, use a solvent with a dielectric constant of 10 or less and or a solvent with a dielectric constant of 15 to 35
If treated with solvents in order of decreasing dielectric constant, the components extracted with these solvents will be removed, reducing the amount of other components mixed in with the active components of the desired hot water extract, and increasing the pharmacological effect. be able to.

The solvent used for extraction in the present invention is for extracting the target component based on the dielectric constant, and the dielectric constant range divided in the present invention is one aspect that is considered to be effective, and further subdivision is possible. It is also possible to convert

Table I below shows typical solvents and their dielectric constants.

Note that the solvents used are not limited to these.

Hereinafter, the anti-neoplastic agent of the present invention will be specifically explained with respect to its extraction example, efficacy test of the extract, effective dosage, efficacy comparison test with existing anticancer agents, formulation, toxicity, etc.

Extraction Example I 100 r/l of benzene was added to dried neem bark ICH9, and extraction was performed for 1 hour with occasional shaking.

Thereafter, 100 ml of benzene was added to the extracted residue through the mouth, and the same operation was repeated, thereby repeating the extraction with benzene three times in total.

100 r/l of methanol was added to this benzene extraction residue, and the same extraction operation as that performed with benzene was performed.

Add 1 liter of room temperature water (deionized water) to this methanol extraction residue.
00ml was added and the same operation as above was repeated.

An additional 100 ml of water was added to the water-extracted residue, and the extraction operation was performed three times in the same manner as above in a boiling water bath.

When these hot water extracts were concentrated in a rotary evaporator, dried and powdered, a dry hot water fraction powder of 270.5''Q was obtained.

Extraction Example ■ 10 g of dried neem bark was subjected to an extraction operation different from Extraction Example ■.

That is, when the extraction treatment with benzene was not performed, ethanol was used instead of methanol, and the other treatment was exactly the same as in Extraction Example I, 364.5 mL was obtained as a hot water fraction.
A dry powder of 1- was obtained.

Extraction Example III To 10 g of dried neem bark was added water and then hot water extraction in the same manner as in the above example, without adding benzene and methanol or ethanol extraction as in the above example, to obtain a hot water fraction. So 421.8”2?
A dry powder of was obtained.

The water used for extraction in the above extraction example is deionized water, but is not limited to this, and can be any water such as tap water, well water, distilled water, salt water, RO water (water obtained using a reverse osmosis membrane), etc. Similar results were obtained using

The temperature of the water is 0 to 40°C, preferably room temperature.

This is because it is better to remove extractable components of water as much as possible.

A test was conducted in which the hot water extract of neem bark obtained as shown in the above example was applied to mouse L-5i78Y cells, Sarcoma 180 ascites cancer, and Sarcoma 180 solid cancer.

The pharmacological efficacy, dosage, and comparative effects with existing anti-convulsants, etc. revealed as a result of the test are explained below.

[Test 1] (Effect on mouse L-5178Y cells) (1) Cell preparation A medium containing 3 lXIO cells/ml of mouse L-5178Ymi cultured for 3 days in RPMI-1640 medium containing 10% fetal bovine serum was prepared. 50 μl per well was added to a U-bottom microplate.

(2) Judgment method 50 μm of the above-mentioned hot water extract sample according to the present invention dissolved in a medium was placed in each hole.

The sample concentration shown in the following table (■) means the final medium concentration.

This was cultured for 2 days in a carbon dioxide incubator at 37°C.

After culturing, cells were collected from each well and counted, and the cell numbers were compared with a control without a sample.

The control cell number was approximately 8°6 x 105 cells/ml.

Similar tests were conducted using mitomycin C (MMC) and pleomycin (BLM) to confirm drug efficacy.

The above test results are shown in Table H below.

In addition, ID50 in the table below means a sample concentration that reduces the concentration to 1/2 of the cell concentration without adding a sample.

(3) Results The test results shown in Table 3 above were obtained for the hot water fraction of the above extraction example and the comparative drug.

Although the medicinal efficacy is not comparable to that of MMC and BLM used as comparison controls, the neem bark hot water extract fraction of the present invention has a small D5o value, and all examples have strong cancer cell growth-inhibiting activity. was confirmed.

Furthermore, it is presumed that the activity of the above-mentioned hot water fraction will be increased if the active ingredient is purified.

[Examination ■]

(Effect on Sarcoma 180 ascites cancer) (1) Sample preparation Phosphate buffered saline (PBS; phosphoric acid 9.5m manufactured by Gibco)
0.5% carboxymethyl cellulose (C
Samples of the hot water extraction fractions shown in Extraction Examples were suspended or dissolved in a solution in which MC'') was suspended to the respective concentrations.

(2) Sarcoma 180 cancer cells transplanted into LCR mice cultured and subcultured in the peritoneal cavity
Cancer cells were taken out along with ascites and diluted appropriately with physiological saline so that the number of cells was 1 x 108 cells/ml.

This cancer cell suspension 0. lll1l is a 4 week old male ICR
It was transplanted into the mouse abdominal cavity using a syringe.

Therefore, the number of transplanted cells per animal was 1×10 7 cells.

(3) Sample administration: From the next day after transplanting mouse Sarcoma 180 cancer cells, once a day for 4 consecutive days, the sample prepared as described in (1) was injected into the abdominal cavity of the mouse by 0.1 m using a syringe.
1 dose was administered.

■ Samples, 6 mice were used per concentration.

In addition, as positive control samples, mitomycin C (MMC), pleomycin (BLM), actinomycin D (ACD), 5-fluorouracil (5-FU
) and su·r clofosamide (CYP).

As a control, the sample was dissolved in the above-mentioned CMC-containing PBS and administered in the same manner.

Dosage is based on mouse body weight IK? Displayed as per weight.

(4) Method for determining effectiveness The weight X of each mouse was measured at the 7th mound after cancer cell transplantation.

Next, after removing the entire amount of ascites accumulated in the abdominal cavity, the weight (Y) of the mouse is measured again, and X-Y is taken as the amount of ascites.

The ascites was sucked into a hematocrit tube and heated to 15,000G at low temperature using a hematocrit measuring rotor.
The cells were centrifuged for 5 minutes, and the asidecrit value (ratio of cells in ascites), which corresponds to the hematocrit value of blood, was measured.

By multiplying the amount of ascites by this value, the volume of cells occupying the total ascites can be determined.

This is calculated as the total cell capacity (TPCvl).
cell volume).

In the control, the total double water volume was 6-10ml, and the TPCV was 1.6-2.
, 5 rnl.

Ratio of TPCV between sample administered and control (T/C)
If this value is 100 to 66%, it has no effect on cancer (@toji), if this value is 65 to 41%, it has an effect of +1 against cancer (1), and if this value is 40 to 11%, it has the same effect (2).
The test results for the neem bark hot water extract sample according to the present invention and a drug sample for comparison are shown in Table 2 below, with those of 10 to Ofb being 3 (++10).

(5) Results The above test results prove that the neem bark hot water extract according to the present invention has high activity, although it is not as good as MMC, BLM, etc. used as comparison targets.

For the hydrothermal fraction (I) in the table above, the minimum effective concentration is the dose - T/
From the C relationship diagram, it was found to be approximately 65 ■/Kp.

In the above tests of the hydrothermal fraction of the present invention, high doses showed some toxicity even to mice, as detailed below.

Although the neem bark extract of the present invention has not yet been sufficiently purified, it exhibits the above-mentioned medicinal effects, and is expected to exhibit even greater efficacy if further purification proceeds.

[For testing] (Effect on Sarcoma 180 solid gun) (1) Sample preparation test H was conducted in the same manner as (1).

(2) Grafting of Sarcoma 180 solid cancer cells A cell suspension of 108 cells/ml was prepared in the same manner as in Test H (2).

Cancer cells were implanted subcutaneously into the back of a 4-week-old male ICR mouse using a syringe using this suspension 01ml1J.

(3) Sample administration test Same as (3) of H, 6 mice were used for each sample concentration.

(4) Effect evaluation method Cancer tissue that had grown to a weight of 15 mouths after cancer cell transplantation was cut out, its weight was measured, and the average value of 6 animals per group was taken.

The effectiveness was determined by calculating the ratio (T/C) of this average weight to that of the control.

The weight of the controls was 1.5-3.5 g.

Those with a ratio value (T/C) of 100 to 71% have no effect on cancer (→ end), those with a ratio of 70 to 51 have an effect of plus 1 on cancer (interest), and those with a ratio of 50 to 21% have the same effect of 2 ( +
The test results for the neem bark hot water extract sample of the present invention and a comparative sample are shown in the table below, with 20% to 0% as 3 (10+).

(5) Results As is clear from the above test results, the hot water extract of neem bark according to the present invention exhibits high activity against Sarcoma 180 solid cancer.

Although it is not as good as MMC, BLM, etc. used for comparison, it exhibits the stated medicinal efficacy even though it is not sufficiently purified, so it is presumed that it will exhibit even more activity if the degree of purification is increased.

Regarding the hot water fraction (I) in the above table, the minimum effective dose was found to be approximately 30711'i/Ky from the dose-T/C relationship diagram.

In addition, in the above test, at high doses, it also showed some toxicity to mice, as will be detailed later.

〔formulation〕

The dry powder of the hot water extract of neem bark obtained as described in the extraction example above is placed in a container such as a vial alone or with a stabilizer, and separately in a container such as an ampoule, distilled water for injection, physiological Prepare saline solution, glucose solution or carboxymethylcellulose (CMC) suspension,
Before use, the powder is suspended and injected.

In addition, it may be made into an emulsion and injected.

For example, a water-in-oil (Wlo) emulsion uses a combination of a mineral oil such as liquid paraffin, a vegetable oil such as sesame oil, and peanut oil, and a surfactant such as sorbicun fatty acid ester.

(1) Formulation Example Extraction Example Dissolve 200 ~ of the hot water extract of neem bark obtained in (■) in 100 ml of sterile physiological saline, and add 1 rI of this solution.
The solution was aseptically dispensed into vials and lyophilized.

In this way, a formulation containing 2 parts of neem bark hot water extract in 1 vial was obtained.

It is sometimes used by dissolving it in distilled water for injection.

[Toxicity]

ICR Mau Mau (body weight 19-21
．． The LD5o value using 0g) was 2 for intraperitoneal administration (ip).
00m? /Kri Tet6 Ivy.

As mentioned above, the neem extract extracted by the method of the present invention exhibits remarkable effects against various malignant neoplasms as demonstrated in the above tests, and is comparable to existing effective anticancer agents. It is expected that further purification and detoxification will lead to the development of an effective anti-malignant neoplastic agent.

[Brief explanation of the drawing]

The accompanying drawing shows I of the hot water extract of neem bark obtained in Example I.
It is an R absorption spectrum diagram.

Claims (1)

[Claims] 1. Neem (Melia azadirachtal)
An anti-malignant neoplastic agent containing as an active ingredient a substance obtained by extracting the bark of Melia azadirachta with water at 0 to 40°C and extracting the extraction residue with hot water at 40 to 100°C. 2. Before extracting neem bark with water at 0 to 40°C, use an organic solvent with a dielectric constant of 10 or less and/or an organic solvent with a dielectric constant of 15 to 15.
The anti-neoplastic agent according to claim 1, wherein the anti-neoplastic agent is extracted with 35 hydrophilic solvents in order of decreasing dielectric constant.