JPH02268120A - Antitumor composition - Google Patents

Abstract

PURPOSE:To obtain a composition useful as an anti-cancer drug, especially a remedy for leukemia, having excellent antitumor activity and low toxicity, comprising a specific polysaccharide compound prepared from root of Panax notogenseng. CONSTITUTION:An antitumor composition which is obtained from root of Panax notogenseng and has the following characteristics. (a) White or light brown powder and slightly water-soluble. (b) Having 3,000-20,000 calculated as polyethylene glycol of molecular weight range and a main peak at 5,000 average molecular weight by gel filtration analysis through high performance liquid chromatography. (c) Having maximum absorption of infrared absorption of KBr tablet approximately at 3,423cm<-1>, 1,636cm<-1> etc., free from ultraviolet absorption in an aqueous solution. (d). Containing a polysaccharide comprising alpha-1,4 glucane, positive in phenol sulfuric acid reaction and negative in Elson- Morgan reaction in qualitative reaction of saccharide. The composition is orally or parenterally administered to human, a dose thereof is 0.01-5g per adult daily and administered once or dividedly 2-3 times.

Description

DETAILED DESCRIPTION OF THE INVENTION "Field of Industrial Application" The present invention relates to novel antitumor compositions obtained from crude drugs. More specifically, the present invention relates to a novel antitumor composition obtained from Panax ginseng [also known as 37 ginseng, 37 ginseng, root of 37 ginseng], which is characterized by having the following physical properties.

(a) White to light brown powder, hardly soluble in water (first
(b) The molecular weight range is from 3,000 to 20,000 in terms of polyethylene glycol (manufactured by Wako Tsumugi Kogyo Co., Ltd.);
High performance liquid chromatography [column, TSK-get
G3000 PWxt, (manufactured by Tosoh Corporation), solvent.

Gel filtration analysis using 0.2 M phosphate buffer (pH 6,9) containing 1/10 volume of acetonitrile showed a main peak at an average molecular weight of 5000. (C) There was no ultraviolet absorption in the aqueous solution, and the infrared absorption of KBr tablets is 34
(d) Contains a polysaccharide consisting of α-1,4 glucan, which has a maximum absorption near 23cm-1, 1636cm-”, and the phenol-sulfuric acid reaction is positive in the qualitative sugar reaction.
The Erzn-Morgan reaction is negative. ``Prior art'' A large number of physiologically active ingredients have been discovered in various herbal medicines, and from 37 to 20 years ago, there are many bioactive ingredients with an average molecular weight of 100,000 or more that have host-mediated antitumor activity. Polysaccharide components have been obtained (JP-A-61-18722, JP-A-61-10)
No. 9732).

"Problems to be Solved by the Invention" The present inventors conducted various studies in order to find a new polysaccharide component having excellent antitumor activity from Sanshichi.

"Means for Solving the Problems" As a result of various studies, the present inventors have discovered that a novel antitumor composition having the above-mentioned physical properties can be obtained from Sanshichi, and have completed the present invention.

The antitumor composition of the present invention can be obtained as follows.

That is, after pulverizing Sanshichi, water or a mixed solvent of water and a water-soluble organic solvent such as ethanol is added, usually 50 to 10
Extract for 0.5-2 hours at 0°C.

The amount of extraction solvent to be used is 2 to 10 times, preferably 3 to 6 times, by weight relative to 37.

After extraction, filtration or centrifugation is performed, and the extraction residue is extracted again in the same manner as above. This operation is repeated as necessary, and the obtained extracts are combined.

Next, the extract is directly or concentrated and subjected to column chromatography packed with a porous polymer such as Diaion HP-20 (manufactured by Mitsubishi Kasei Corporation), and eluted with at least 3 times the volume of water relative to the carrier capacity of the column. do. An extract powder is obtained by concentrating the solvent under reduced pressure and drying under reduced pressure, spray drying, or freeze drying. The saponins contained in Sanshichi can be removed by the above operations.

The above extract powder is dissolved in hot water and subjected to molecular weight fractionation treatment such as gel filtration, ultrafiltration or dialysis, so that the molecular weight range is 3000 to 2000 in terms of polyethylene glycol.
The antitumor composition of the present invention can be obtained by obtaining both fractions of 0 or less, concentrating the solvent under reduced pressure, and drying under reduced pressure, spray drying, or freeze drying.

In the above-mentioned gel filtration, ultrafiltration or dialysis, for example, Sephadex G-100 (manufactured by Pharmacia), ultrafiltration membrane IF-2C3-10PS (manufactured by Tosoh), etc.
Alternatively, cellulose dialysis membrane Spectrabore 6 (MWCO)
1000) (manufactured by Wako Tsumugi Kogyo Co., Ltd.).

The antitumor composition of the present invention exhibits antitumor activity by directly inhibiting the uptake of uridine into human leukemia cells, as shown in Test Examples below, and also has low toxicity.

The antitumor composition of the present invention can be prepared into solid preparations such as powders, granules, tablets, or capsules, or liquid preparations such as injections, using conventional pharmaceutical additives, and administered orally to humans. Or it can be administered parenterally. The dosage varies depending on the patient's condition, age, weight, etc.
Usually, for adults, 0.01 to 5 g per day may be administered at once or in 2 to 3 divided doses.

"Actions and Effects of the Invention" From the test results shown below, the antitumor composition of the present invention has excellent antitumor activity and low toxicity, and is useful as an anticancer agent, particularly as a therapeutic agent for leukemia.

Test Example 1 (Anti-tumor activity) Anti-tumor activity was investigated using as an index the rate of inhibition of the uptake of uridine, which is one of the pyrimidine nucleosides, into cultured human leukemia cells (human lymphoid leukemia cell line NALM-18).

(1) Sample/powder of Example 1 (antitumor composition of the present invention'&J) (2
) Prepare an aqueous solution (10 mg/ml) of the powder of Test Method Example 1,
The sample solution was sterilized in an autoclave for 15 minutes.

RPMI-1640 medium (manufactured by Wako Tsumugi Kogyo Co., Ltd.) containing 10% fetal bovine serum was added to the human lymphoid leukemia cell line NALM-18 (Jichi Medical University) to adjust the cell number to 5 x 10 S cells/ml. Then, 3 ml each was dispensed into a 12-well tissue culture plate. 30g of the above sample solution in each well
1 and mix well, heat the plate at 37°C15
The cells were cultured for 24 hours under %GO□ conditions, and 1 ml was dispensed into test tubes.

[3H]-uridine aqueous solution (0,1
After adding 10g1 (mC1/ml) and mixing well, culture was performed again at 37°C for 2 hours under 15% CO2, and then incubated at 4°C.
Centrifugation at 1200 rpm for 10 min. After removing the upper groove and adding 25% albumin aqueous solution to the obtained pellet and mixing well, 2 ml of 5% trichloroacetic acid aqueous solution containing 0.05 M sodium pyrophosphate was added, and centrifuged at 4 °C for 10 minutes. .

1200 rpm) L/ta. Discard the supernatant and repeat the same procedure 2.
Repeatedly, a precipitate was obtained. 200 liters of the precipitate
After dissolving in Olene-350 (manufactured by Packard), the solution was incubated overnight, a toluene scintillator was added, and the solution was taken up into cells using a liquid scintillation counter.
3H couridine amount [S (dpm/10'cell)
] was measured. Next, as a control, [3H] taken into cells when distilled water was used instead of the sample solution.
- The amount of uridine [C (dpm/10'cell)] was measured in the same manner as above.

The inhibition rate of 3H caulidine uptake into the cells was calculated using the following formula.

Uptake inhibition rate (%) = (at 1-) (3) Test results The test results are shown in Table 1.

Table 1 x 100 Test Example 2 (Acute Toxicity) The powder of Example 1 (anti-tumor composition of the present invention) was suspended in water (concentration, 30 mg/ml) per liter of ICR male mice (5 days old). target, weight 29-30 g, - group 10)
00 mg/kg was administered intraperitoneally, and the number of deaths was observed for up to 2 weeks after administration. As a result, no deaths were observed, and the LD value of the powder was 1000 mg/kg or more.

"Example" Next, the present invention will be explained by giving examples.

Example 1 After pulverizing 2 kg of Sanshichi, 10 l of water was added and extracted at 70°C for 1 hour. After extraction, it was filtered, and the extraction residue was extracted again in the same manner as above, and the obtained extracts were combined. Then,
The extract was subjected to column chromatography (column 9cm 5d) packed with Diaion HP-20 (manufactured by Mitsubishi Kasei Corporation).
x 35 cm) and flow rate 500 m1/hr), water 81
It was eluted. The solvent was concentrated under reduced pressure and lyophilized to obtain 268 g of extract powder (yield: 13.4%).

The above extract powder was dissolved in 151 of water under heating, and the filtrate was divided into 31 portions into 5 portions using an ultrafiltration membrane UP"2C3-10PS (manufactured by Tosoh Corporation) at a flow rate of 11/min until the filtrate was 2.71 g.
It was cycled until. The obtained filtrate was combined and the solvent was concentrated under reduced pressure to obtain 1.351 filtrate.

Next, the filtrate was passed through a cellulose dialysis membrane Spectrabore 6.
(MWCO1000) (manufactured by Wako Tsumugi Kogyo Co., Ltd.) 3 bottles (4,
Dialysis was performed for 12 hours against distilled water (61 cm) using a 5 cm φ x 50 cm filter. The external dialysis solution was discarded, distilled water 61 was further added, and the same dialysis operation was repeated six times.
) was obtained.

The antitumor composition of the present invention exhibited the following physical properties.

(a) White to light brown powder, hardly soluble in water (first
(b) The molecular weight range is from 3,000 to 20,000 in terms of polyethylene glycol (manufactured by Wako Tsumugi Kogyo Co., Ltd.);
High performance liquid chromatography [column, TSK-gel
G3000 PWxt, (manufactured by Tosoh Corporation), solvent.

Gel filtration analysis using 0.2 M phosphate buffer (pH 6,9) containing 1/10 volume of acetonitrile showed a main peak at an average molecular weight of 5000 (c) There was no ultraviolet absorption in the aqueous solution, but infrared absorption of the KBr tablet. is 34
(d) In the qualitative reaction of sugars, the phenol-sulfuric acid reaction is positive and the Elson-Morgan reaction is negative. Thin layer chromatography after hydrolysis with 2N hydrochloric acid 13C-NMR (75MHz, solvent, D20, δppm
), the signals attributed to α-1,4-gluen bonds [102,3 (glucose C□), 79.5 (glucose C,), 76.0 (glucose C3), 74.
2 (glucose C2), 72.8 (glucose C,)
, 63.2 (glucose C6), etc.] are observed.

Claims (2)

[Claims] (1) An antitumor composition characterized by having the following physical properties: (a) white to light brown powder, hardly soluble in water; (b) molecular weight range of 30% in terms of polyethylene glycol.
00 to 20,000, and the average molecular weight is 5,000 as determined by gel filtration analysis using high performance liquid chromatography.
(c) There is no ultraviolet absorption in an aqueous solution, and the infrared absorption of KBr tablets is 3423 cm^-^1 and 1636 cm^
(d) Contains a polysaccharide consisting of α-1,4 glucan, which has a maximum absorption near -^1, and the phenol-sulfuric acid reaction is positive in the qualitative reaction of sugars.
Elson-Morgan reaction is negative (2) Antitumor composition according to item 1 obtained from No. 37