JPS6281320A - Carcinostatic agent containing lambda-spirulinan - Google Patents

Abstract

PURPOSE:To provide a carcinostatic agent containing lambda-spirulinan which is a sugar produced by an algae Spirulina subsalsa. CONSTITUTION:The objective carcinostatic agent can be produced by using a polysaccharide lambda-spirulinan at least containing rhamnose, mannose, uronic acid and N-acetyl sugar as constituent components and produced by Spiruline. The agent has low side effect and strong carcinostatic activity. The lambda-spirulinan used as an active component is soluble in an aqueous solution of potassium chloride and can be derived from a spirulinan composed of lambda-spirulinan and kappa-spirulinan which is insoluble in an aqueous solution of potassium chloride. It is preferably administered parenterally (e.g. pertoneal infusion, intravenous injection, intramuscular injection, rectal infusion, etc.). Dose: 20-0.01mg/kg daily, preferably 2-0.1mg/kg daily. It can be administered for a long period, every 1-5 days.

Description

[Detailed Description of the Invention] [Industrial Application Field] This invention is directed to the use of algae Spirulina subsalsa (Sp
A new IJ produced by Irulina 5ubsalsa)! The present invention relates to an anticancer agent containing λ-spirulinan as an active ingredient.

[Conventional technology]

It is already known that certain poly[T]s have anticancer effects. However, λ-spirulinan, which is the active ingredient of this invention, was first announced at the 1980 Japanese Society of Agricultural Chemistry Conference, and it is completely unknown that this spirulinan has anticancer effects.

JP-A-54-73108 describes that Spirulina platensis or its extract has an anticancer effect. However, there is no description of the physical characteristics of the active ingredient that provides the anticancer effect, and there is no suggestion that Spirulina subsalsa has an anticancer effect.

[Problem that the invention seeks to solve]

Conventional anticancer drugs containing polytjj as active ingredients have the advantage of having fewer side effects than other types of anticancer drugs, but on the other hand, their efficacy as anticancer drugs has not always been satisfactory. The present invention aims to provide a multi-IJgLrT type anticancer agent with few side effects and strong anticancer activity.

[Means for solving problems]

The problem mentioned above is that the constituent components (rhamnose, fucose, xylose, galactose, glucose,
The solution is an anticancer drug comprising the polysaccharide λ-spirulinane, which comprises mannose, uronic acid and N-acetyl sugar and is produced by Spirulina.

An anticancer agent containing a mixture of λ-spirulinan and monospirulinan as an active ingredient is described in Japanese Patent Application No. 60-006668. As a result of a more detailed study of the active ingredients of the above-mentioned anticancer agents, the present inventors found that 3MKCl-soluble λ-svirulinan has a stronger anticancer effect than a mixture of λ-spirulinan and monosvirulinan (spirulinan mixture). It was discovered that it has a cancer effect, and based on this knowledge, the present invention was completed.

1. Active ingredient The active ingredient of the anticancer agent of the present invention is λ-spirulinan.

Svirulinan consists of λ-type spirulinan that is soluble in an aqueous potassium chloride solution and λ-type spirulinan that is insoluble in an aqueous potassium chloride solution. Method for producing spirulinan and λ-
A method for separating type 1 spirulinan from type 1 subvirulinan is described in detail in Japanese Patent Application No. 152147/1982, and specific examples thereof are described in Reference Examples I and 2 of the present specification.

Spirulinan has the following physical and chemical properties.

■ Elemental analysis and ash content The results of elemental analysis and ash content are shown in Table 1 below.

■ Detection reaction The results of various detection reactions are shown in Table 2 below.

+: Positive -: Negative As is clear from Table 2, Yaphe's resorcinol reaction does not show a clear positive result, and the spirulinan of this invention does not contain 3,6-anhydrogalactose, or even if it does, it does not contain 3,6-anhydrogalactose. The amount is extremely small and clearly different from carrageenan.

This is consistent with the results of hydrolyzing subirurinan with IN hydrochloric acid for 2 hours and examining its constituent components by paper chromatography.

In addition, the carbazole-sulfuric acid reaction, which is a qualitative test for uronic acid, and the Morgan-Elson reaction, which is a qualitative test for N-acetylhexosamine, were both positive, indicating that spirulinane is a uronic acid and N-acetylhexosamine.
- Estimated to contain acetylhexosamine.

Furthermore, since the Dodgson reaction was positive, it was confirmed that spirulinan contains a sulfate group.

(2) IR spectrum The IR spectrum is shown in FIG. In this figure, 1
C of aldehyde group or carboxylic acid near 725 cm-'
Absorption originating from the =0 group is seen. Carboxylic acid C=O
The presence of spirulinane and λ- in the form of potassium salt
It is also estimated by the appearance of absorption near 1610C1ll-' in spirulinan.

Absorption of acetamide near 1660 cm-' and 1530c+n-', 1450 cm-' and 1400 cm-'
Absorption derived from methyl groups can be seen nearby. 1240c
Absorption derived from sulfate ester (S=0) group in m-', 8
Absorption derived from the C-0-S group is seen at 20 cm-' and 840 cm-', indicating the presence of sulfuric ester. 8
The absorption at 20 cm-' indicates the presence of equatorially bound 6-sulfate, and the absorption at 840 cm-' indicates the presence of axially bound 4-sulfate. There was a slight absorption at 940 cm-' indicating the presence of 3.6-anhydrogalactose.

The above IR spectrum results agree well with the detection results described above.

(2) The constituent sugar subirurinan was converted into trimethylsilate, and the hydrolyzate thereof was analyzed by force chromatography, and the results shown in Table 3 below were obtained.

Below are the margins Chromatography conditions Column: 2mX3ms, 3% 0V-225, Gas Chrome Q; Pour temperature: 230°C; Column temperature: 140°C; Carrier gas (N2) flow rate: 50m7! /min; Equipment: Shimadzu GG 7 AG As is clear from Table 3, spirulinan contains a small amount (rhamnose, fucose, xylose, galactose, glycose, and mannose, and is expected to contain other sugars as well. Ru.

Note that the presence of galactose and glucose was also confirmed by tests using the respective oxidases.

■ Gel forming property The subirrinan of this invention is K", NHa".

Or form a gel in the presence of Ca*+.

The mucilage polysaccharide having the above properties has not been confirmed to exist in Spirulina platensis (Spirulina platensis) or other blue-green algae, and is a novel mucilage polysaccharide.

(2) Pharmacological properties Antitumor properties were tested using 6-week-old female mice [5 mice in each CR group]. All mice were intraperitoneally inoculated with 1×10 6 Sarcoma 180 cells, and the subsequent survival days were measured. The test material λ-spirulinan was dissolved in physiological saline and administered intraperitoneally 10 times, once a day from the first to fifth day and from the seventh to the 14th day. In the control group, only physiological saline was administered. The single doses for the test group were 50 mg/kg, 20 mg/kg and 2 mg/kg.
It was set as g.

The results are shown in Table 4 below.

Note (11 Life prolonging effect) As is clear from Table 4, the anticancer agent of this invention was administered to monkey macaques in mice at a daily dose of 50 mg/day.
showed a life-prolonging effect at ~2 mg/kg.50
mg/kg to 10 mg/+r showed a significant survival effect.

The effect of a spirulinan mixture consisting of λ-spirulinan and monosvirulinan was tested by the same method as above,
The effect was compared with that of λ-svirulinan described above. Svirulinan mixture is 20mg/kg/day (total 200mg/day)
kg), and 50 mg/kg 7 days (total 50
The survival effect at each dose was determined and compared with that of λ-svirulinan.

In addition, 20 mg/kg of λ-spirulinan (total 20
IOB/kg/day (total 100,111 g/k+r)
It was calculated from the results at mg/kg/day (total 500 mg/kg). The results are shown in Table 5.

Margin below 1st L-table The results are shown in FIG.

As described above, the λ-svirulinan of the present invention showed a higher life-prolonging effect than the spirulinan mixture.

(3) An acute toxicity test was conducted using 6-week-old female C3H mice. A solution containing 5.5 mg/ml of λ-spirulinan was prepared, and 0.4 mβ of this solution was intraperitoneally administered 5 times at 45-minute intervals on the first day, and 4 times in the same manner on the second day. , the total dose was 1000 mg/kg. The changes in the average body weight of mice were as follows.

Time (hour) 0 24 48 72 After 24 hours, temporary diarrhea symptoms were exhibited, but the animals quickly recovered, and all animals were alive 72 hours after the end of administration.

As described above, the anticancer agent of the present invention has a high anticancer effect and is hardly toxic.

The anticancer agent of the present invention is effective against various cancers, and is preferably administered parenterally, such as intraperitoneally, intravenously, intramuscularly, or enterally, such as rectally. The effective dose in mammals including humans varies depending on the type of cancer, patient symptoms, etc., but is approximately 20 mg/kg to 0,000 OIB/kg per day, preferably 2 m
g7 kg to 0.1 mg/kg. This medicament can be administered over an extended period of time every 1 to 5 days.

(2) The anticancer drug of the invention of preparation can be in the form of a preparation commonly used as a parenteral administration agent, such as an intravenous injection, an intravenous injection, an intraperitoneal injection, etc. Specifically, for example, the active ingredient is Aqueous λ-spirulinan? '8? (9) or an aqueous suspension. The base for this formulation is preferably an isotonic solution, such as physiological saline. The medicament may further contain an injectable buffer. , a solubilizer, a surfactant, a preservative, etc. can be added.In addition, these solutions or suspensions can be freeze-dried to form a powder preparation.In this case, sterile water should be added before use. It is used as a solution or suspension by adding

These drugs can be manufactured according to conventional pharmaceutical methods.

〔Example〕

Next, in order to explain the present invention more specifically, examples will be described regarding the manufacturing method and formulation method of subirurinan, which is an active ingredient.

1 good!　Spill 1　　+l“notification A medium having the following composition was prepared.

Below margin NaHCOz 16.0 g; Mg5On 0.2
g ; A5 solution 1.0 m 1 ; KzllPOt
O,5; CaC1z O,04; Hzo
1. OI! ;NaNOx 2.5:
FeSO40,01; NaC11,0; EVT
A, 0.08; (*) A5 solution HJO32,86g; MnC1z・4Hz0
1.81g; Zn5Oa '7HzOO, 22;
CuSO4・5Ht0 0.08; NazMO
40,021: 1 drop of Conc-HzSOa; Ih
0 1.0 1: This medium 100
M1 was inoculated with Spirulina subsalsa and heated to 37℃ under 4000 lux fluorescent lamp irradiation without carbon dioxide aeration.
Cultured at 40°C. It was confirmed that mucilage polysaccharide was secreted outside the algae during the culture process. Under these conditions, this strain grew by elongating filamentous algal cells and floated to the surface of the liquid. It formed a compact spiral filament compared to Spirulina platensis.

Wash algae with water, add 80% methanol, 80% acetone, 1
The dye was removed by boiling with 0.00% ethanol and ether. The algal bodies were collected and the spirulinan was extracted by heating at 90° C. for 1 hour in an aqueous solution containing 0.2% sodium chloride and 0.1% sodium bicarbonate, and filtered.

Spirulinan was precipitated by adding cetyltrimethylammonium bromide (Cetabron) to this spirulinan extract at a concentration of 2%. This precipitate is 8
White spirulinan 44m was obtained by washing with 0% ethanol-1100% ethanol and ether and drying.
I got g.

0.1 g of spirulinan produced according to the method described in Reference Example 1 was heated to 90°C with vigorous stirring.
．． Dissolved in distilled water to a concentration of 1% and filtered. To the filtrate was added 0.1 volume of 3M potassium chloride solution, the solution was mixed and cooled in a crushed ice bath for 1 hour. Centrifugation at 10,000 rpm for 1 hour at 50°C;
It was divided into an insoluble fraction and a soluble fraction.

The insoluble fraction was resuspended in 0.3 M sodium chloride solution, centrifuged, the chlorine was removed by washing the insoluble material with 80% warm ethanol, and 100% ethanol, acetone and ether; After drying, 0.03 g of Spirulina was obtained.

On the other hand, the soluble fraction was precipitated by adding 2.5 volumes of 100% ethanol, washed with 80% ethanol, 100% ethanol, acetone, and ether, and dried;
0.069 g of λ-spirulinan was obtained.

The ratio of λ-spirulinan to -spirulinan is approximately 69
．． 1:30.9.

Implementation ■ Top Next - M (13 Injection solution λ-Spirulinan 5g Sodium chloride 4.5g Sucrose 25g Dissolve all ingredients in 500m!! of distilled water, filter aseptically,
Fill into vials.

(2) Suspension for injection λ-Spirulinan 2g Sodium chloride 0.15g Glycerin
2 g All ingredients are dissolved in 20 mA distilled water, sterile filtered and filled into ampoules.

[Brief explanation of drawings]

FIG. 1 shows the rR spectrum of spirulinan. FIG. 2 is a graph showing a comparison of the anticancer effects of λ-spirulinan and a spirulinan mixture.

Claims (1)

[Claims] 1. At least rhamnose, fucose,
xylose, galactose, glucose, mannose,
Spirulina contains uronic acid and N-acetyl sugar.
Subsalsa (Spirulina subsalsa)
An anticancer agent comprising the polysaccharide λ-spirulinan produced by.