JPS5853760A - Screening of antitumor polysaccharide - Google Patents

Abstract

PURPOSE:To achieve the screening of an antitumor polysaccharide simply and quickly, by bringing a diluted polysaccharide solution not containing endotoxin into contact with a hemocyte extract of a horseshoe crab. CONSTITUTION:This screening of a polysaccharide having an antitumor effect is characterized by selection of a polysaccharide indicating positivity in a Limulus test by bringing a diluted polysaccharide solution not substantially containing endotoxin into contact with a hemocyte extract of a horseshoe crab. Applicable polysaccharide includes those obtained from animals, higher plants and lichen, those obtained from bodies and culture solutions of mildew, bacteria and yeast and additionally, those obtained by oxidation, reduction or hydrolysis of these polysaccharides and those obtained by the hydroxyethylation and carboxymethylation thereof. The measurement of the positivity in the Limulus test is done by determining the extent of gelation of the hemocyte extract of the horseshoe crab or the activity of a coagulated enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention uses horseshoe crab O* bulb extract to produce antitumor polysaccharide O.
Relates to a method of screening.

Antitumor polysaccharides are included in so-called immunostimulants that increase host resistance and suppress tumor growth, and they have far less side effects than various chemotherapy drugs. Its usefulness in avoiding the new year is attracting more and more attention. Toremo O anti-tumor polysaccharides are widely distributed in nature), for example V
Hiro Lentinan from Iruke, Pakan Mafun from Yarei,
Kawora Takekara is Krestin, Sue and Ichitake are V.
Zotsuifun has been obtained from yeast, Zairokuzan has been obtained from yeast, hemicellulose has been obtained from wheat straw, and GK-3 has been obtained from lichen. Some of these have already been highly evaluated as cancer immunotherapeutic agents, and some are currently under development as anticancer agents, but round-O research to find even more effective antitumor polysaccharides (widely It is actively carried out.

The measurement of the anti-inflammatory effect of these O polysaccharides is usually carried out using Ma? XK ported and nine ρ coma 180. Ehrlich Calt Norma,
The inhibitory effect of polysaccharides on CCM adenocarcinoma, etc., has been investigated by and, but this method requires a large number of experimental animals and special AT techniques, and on the other hand, it takes one month to determine the effectiveness. %0II- Since it requires a lot of time, it requires a huge amount of expense and a lot of effort.

On the other hand, λIL Johns 1lopk
It was discovered by Tsuru Iocho et al. 98325 (111g6)) that the capto-tr-ojk solution gelled in the presence of endotoxin, and it became widely used. ) to detect endotoxins (by endotoxins)
The stiffness of the resulting gel can be determined by tilting the test tube, and the amidase activity of the coagulation III enzyme, which is activated from an inactive precursor in the presence of endotoxin and is involved in Ogglutination, has been investigated. There is a method of measuring using a synthetic substrate.

The present inventors found that various anti-tumor polysaccharide preparations that are not contaminated with endotoxins strongly gelled horseshoe crab extracts, and after conducting various studies, they found that this 0-gluation is the identification of anti-tumor polysaccharides. 0 occurrence ζ within the concentration range), this IIm! It was recognized that at concentrations other than 1%/%, and that such a unique gelation pattern was observed in polysaccharide K11, which has an antitumor effect, the present invention was completed as a result of intensive studies.

The present invention provides a hemocyte extract of Kabutoga Kl! A polysaccharide obtained from Starier lichen, a polysaccharide having an antitumor effect, characterized by contacting with a polysaccharide dilute solution that does not contain endotoxin, and selecting a polysaccharide that shows a positive Rimuyas test.
Polysaccharides obtained from the cells and culture fluids of molds, microorganisms, and yeasts, as well as polysaccharides obtained by oxidizing, reducing, or hydrolyzing these polysaccharides, as well as polysaccharides obtained by oxidation, reduction, or hydrolysis, ethylation, carboxymethylation, etc. It includes t-t obtained by ethylation. Moreover, these polysaccharides can be applied and used regardless of their solubility in water.

The zero concentration of polysaccharide in the diluted polysaccharide solution varies depending on the polysaccharide, but it is usually about 2 x 10-"11/dFJhb 2 x 1
0-'q/dO concentration 0% may be prepared. As a polysaccharide dilute solution, an aqueous solution is used in cases where the polysaccharide is soluble in water, and an aqueous solution is used in cases where the polysaccharide is not dissolved in water.
Just use a cloudy solution.

The polysaccharide dilute solution used in the present invention needs to contain 110% of foreign materials. ! J purchase end) (
"Fun" means that the polysaccharide sample to be tested has a polysaccharide content of 311 ppm or less, more preferably 0. l
ppm Sakashita refers to the following.

To ensure that the polysaccharide sample is substantially free of endotoxins, dissolve the polysaccharide sample in a sodium hydroxide solution of 2 MI/l, suspend it, and leave it at 4°C overnight to remove endotoxins. What is necessary is to perform a V-coating process.

As the horseshoe crab that provides the hemocyte extract of the horseshoe crab that can be used in the rim ν-less test, any crab that belongs to the taxonomic classification (1. For example, 4 trld@ntatus from Japan
, American OLim 1 mountain trail polyp) leans e Carolnoaoor from Southeast Asia 1 txs 1 prisoner ■ 4 μ discussion, eyes V ha hanging [overturned,! Ridge and powder μis tr
id@ntat■ etc. can be used. Furthermore, as a blood cell extract, an extract obtained by collecting blood from turnips and treating the blood cells obtained under hypotonic conditions can be used directly. ), Pyrot 11
f Cape Cod
oal Company (USA)], Pyrodiene)
(Pyrog@nt) (Mallnokrodt)
, InaI (USA)], Pyrot axis) (Difoo Iabe-ratories)
(USA) jll), Miorobiolog1
calAsscxriat@s (USA) Limulus amoebot zyzeit (LAL) may also be used, but it is generally convenient to dissolve and use a commercially available preparation for endotoxin detection. be.

A positive Limulus test can be determined by measuring the degree of gelatinization of the horseshoe crab blood cell extract or by measuring the OH property of a coagulating enzyme.

In the method of the present invention, in order to measure the degree of gelation when the anti-tumor polysaccharide and horseshoe crab blood cell extract are combined in summer,
(1) Method of determining the hardness of Gefi/ in a southern limit, (8)
After gelation, the amount of protein is usually determined using a protein assay method such as Lawry.
(S) A method of measuring the increase in turbidity O due to gelation using a nephelometer. Regardless of the method used to measure uptake, the polysaccharide is dissolved or suspended in sodium oxide, left overnight at 4°C, and then further diluted to obtain polysaccharide dilutions of various concentrations. Preparing the solution * (g) * Dissolve the Boletus crab extract in distilled water for pharmacopoeial injections. (3) Add 0.1 d of polysaccharide dilute solution of various concentrations and 0.1 mK of turnip/crab blood cell extract-containing solution and mix gently. (4) Seal the container containing the reaction solution O with puff film, and then leave it at 37°C for 1 hour, and then leave it at room temperature for 5 minutes. (5) Tilt the container by 45"K and judge the degree of gelation from the southern limit as follows.

+: Forms a solid gel and the gel does not collapse even if the container is tilted.

+: Forms a gel, but when the container is tilted, the lumps) move.

±: Forms a soft or semi-fluid gel, and flows smoothly.

m: Fluid 11 with almost no change.

A positive limulus test indicates a degree of gelatinization above that of a professional in this Nikkuma courtroom law.

te, Anti-tumor polysaccharide O horseshoe crab hemocyte extract gel & [The acid part and the activation of coagulation enzyme precursor to coagulation enzyme are closely related.
Regarding I, instead of measuring gelation-i), the activity of the coagulating enzyme produced in the summer liquid may be directly measured. As a substrate for this coagulating enzyme, the peptide was made to use NOC nitroaniline (plA)! 11(le-Glu-
Gly-mrg-pHm, Bm-ma4l-Gly-mr
g-p seal, 1loo-Leu-Gly-mrg-
plm, Boo-get(OBg)-Gly-mr
g-p mark, Boo-ma&1-L@u-Gly-mrg-p11m and l1oo-maal-4@r-G1
71 mu rg-plA etc. can be used. We also leave the amino acid sequences of these substrates and the chromophore plA! It can also be used as a substrate by substituting 1-amino-4-methyltamarin (Musou C) with one-party peptide P1 as a substrate, and sometimes using free peptide Plum as a substrate ( The amount of P summer can be measured by the absorbance at 405 nN.
The peptide may be measured by replacing A with a red azo dye.
For example, using a chromogenic synthetic substrate and measuring the MCO concentration using MC as a substrate, the MCO concentration can be measured in the same manner.
7, 183 (1978)), (1) turnip) crab blood cell extract was added to 0.4M)'jnu hydrochloric acid-0. ,04MMg
Dissolve C1g buffer (pl g, O)O, SdK.徊) 1loo-Leu-Ql, a chromogenic synthetic substrate 01
y-murg-pHmu-HC1 is dissolved in distilled water for injection to a concentration of 1.84/sr. (3) Add 0.025 dTh of the horseshoe crab hemocyte extract-containing solution and 0.0251 ml of the synthetic substrate solution to 0.05 ml of the diluted polysaccharide solutions of various concentrations described above, and incubate at 3γ°C for 30 minutes. (4) After reaction 12.
The reaction was stopped by adding 9.4 ag of 5XO acetic acid,
5 Measure the absorbance (mu 40.) of all K>.

In this method, showing positive Limulstes means showing a value of Mu405 of about O,OS or more.

In the above KTh, the abbreviations for amino acids, peptides, and protecting groups are IuPmuC-(tie CComm1gm1
O O! 11Siologloal Iomeuola
It is based on the abbreviation number tur* or the abbreviation commonly used in the public funds concerned. Examples of these are listed below. For amino acids, etc., when optical isomers exist (To), unless otherwise specified, %O indicates the L-isomer.

11. : Iso cost i Vn Gin : Glutamic acid oxy : Gusa V nmu rg2 arginyl ma&1 : Padan 1a@u : WiVn3・r; Sehen 1z: Peny voice Boo : Tar V'r9 -1 Toki V force〃Boe)L
7. In this way, since it shows a positive result in the polysaccharide test that has an antitumor effect, it is possible to eliminate the polysaccharide that has an antitumor effect out of the polysaccharide O that is the test sample. On the other hand, 1i polysaccharide and V-clifostamide (ali
10-7 to 1o-1
q/b shows neither gelation ability nor quasi-solid enzyme precursor activation ability.

In the method of the present invention, in vitro reactions can be carried out using horseshoe crab hemocyte extract seed crystals, so antitumor polysaccharide can be easily and quickly produced in a normal laboratory without using special techniques. - Able to carry out two operations. In addition, this method can save a great deal of expense and labor required for purchasing and breeding animals, and can provide highly accurate results, compared to starlining using experimental animals. ′
The method of the present invention will be explained in detail below using Examples, but the present invention is not limited thereto.

In addition, when carrying out method O of the present invention, it is necessary to sterilize all instruments, reagents, II agents, etc. that will come into contact with the optoganilum bulb extract in advance, and to carry out the de-endolysis in Kugami). It's no good.

Specifically, for example, the gaff device is first thoroughly washed with a neutral detergent, then tap water, and ion-exchanged water, and finally washed with sterile distilled water that does not contain chemicals, and then heated at 180°C for 3 days.
Heat sterilize at 250°C for 30 minutes. Pharmacopoeial distilled water for injection as a good solvent 1 Pharmacopoeia physiological saline can be used as is.

Example 1 guρky (P 8 ) (Cancer Rea 3
8.379 (1978), special meeting 1973-32673. British Patent No. 1352938, Japanese Patent Application Publication No. 53-66442)
Dissolve in KO01 Summer 1aO1 to give 2WjI/mt, leave it overnight, 0.4M-)1S-HCl-04
After diluting 10 times with 1 g of MMgC loose gastric juice (pH 8,0), further diluting with pharmacopoeia saline to obtain various O concentrations.
Prepare ES liquid.

w), Japanese turnip) Derege ρ containing t=0 blood cell extract (
Add 0.3 d of the contents of the ampule of 0.3 d of the above 0.4 M) cis-wood citric acid-0,0
Dissolved in 4MMgClg buffer (pH 8,0) (solution containing blood cell extract).

c), synthetic substrate 11oo-Leu-Gly-mrg-p
HCl was dissolved in distilled water for injection to a concentration of 1.61 RI/d.

;), ps solutions of nine various concentrations prepared in a), O, OSd,
Blood cell extract containing solution 0.025sf and synthetic substrate solution 0
．． 0.1 d of the reaction solution consisting of 0.0255 g was placed in a sterile test tube.
The reaction was carried out at 37°C for 30 minutes. After the reaction, 0.4 d of 12.5% acetic acid was added to stop the reaction, and the absorbance at 405 nm (mu4o5) was measured to give the results in Table 10.

Table 1 ps concentration Cl1l/d) 10-710''310-'
10-'10-'10-" 10-1mu4o5
QDOCLOI Q10 QJII (L91
G330D3 table IK shown tlK, P8 is IO"-'>
! It showed a strong coagulation enzyme precursor activation ability at 10-'W/d.

Example λ - Anti-tumor 1IIll&/bokiV methyl β-1,3-glucan (C
M P 8 ) (IuroPJ, Canoer 1
5.211 (1979).

KO'd JP-A-53-66442) to become 21e/d.
, 1 in CMP8 aoII, left overnight, and then diluted in the same manner as in Example 1 to prepare CMP8 solutions of various concentrations.

), dilute the contents of the ampoule with distilled water for injection at 0.1
I understood sfK*.

0.1 d of the CMP8 solution prepared in step (c) and various concentrations was added to the pregel amplifier p in a) and gently warmed.

2) Seal the ampoule with a paraphrase 904b, 37
Let stand at ℃ for 1 hour and then at room temperature for S minutes.

Tilt the ampoule at 45° and judge the degree of gelation.

4+: A hard gel is formed and the gel Oy# does not collapse even if the bowl is tilted.

＋Nigeρ is formed, but when the ampoule is tilted, it moves as a lump).

±: Forms a soft semi-fluid gel and flows when tilted.

m: There is almost no change in the fluid state.

Table 2 Gel strength -± ++ 廿 + ---j5K and CMFg shown in Table 2 are 10-'>! bi10-'W
Shows strong gel-forming ability with II/Id.

Example 3 As5sooiat@s of Cap
e Manufactured by CodInn, sold by Wako Pure Chemical Industries, Ltd.) O
One sample measurement amount was 0.3 dOO, 4 M) IX-jj [acid-
o.

4MMgC] 41111 solution (pH 8,0) K dissolved (
blood cells and blood cells).

The synthetic substrate Boo-Leu-Gly-mrg-pH-HC'i was injected at 1.6 MI/d.
Dissolved in 1ir water.

C), OMF51 of various concentrations prepared in Example λ @ wave 0
．． A reaction solution of 0.05aF, 0.025wt of a blood cell extraction solution and 0.025d of a synthetic substrate solution was reacted at 37°C for 30 minutes in a sterile test tube.

After the second summer, Mu405 was concubined and treated in the same manner as in Example.

Table 3 As shown in Table 3, CMFg is
If you use a ball extraction proboscis to make a round, you can also use a Japanese turnip round.

KO, 111&O! Dissolved in I, -
The table device was diluted in the same manner as in Example 1 to obtain various concentrations.
Prepare a V-stin solution.

Iχ Rhodotolula glutlnlg I
P 01 125 shares (I? Latitude For
Periaentatlon 0saka Li5t
ofCulture81978 jixth 511
tlon K) in a 200sr Erlenmeyer flask.3. O%.

Yeast extract XQ, 3X e (NH4)5HHPO4G
-1sx, KH2PO40.1%, Mg804-
7120 0.05%, pH 6,8G liquid medium 50mg
K inoculated and cultured with rotary shaking at 2g℃ for 3 days.
11 Spring, 5 d each was transferred to a triangular glass tube and cultured with rotational shaking at 25° C. for 4 days to obtain a culture solution 2.51.

iv) The culture solution was centrifuged, and 20.3 g of sodium acetate was added to 2.47 g of the resulting centrifuged supernatant, followed by 2.47 g of ethanol. The resulting precipitate was collected by centrifugation, dissolved in 500ml of water, and 4.1g of sodium acetate was added followed by 811ml of ethanol. This procedure was repeated twice with the obtained precipitate, and the precipitate was washed with ethanol and dried to obtain 2.4 g of polysaccharide 1125. Polysaccharide 11250 sugar content is phenol-
pssx was calculated by the sulfuric acid method.

The polysaccharide 1125 was measured using the method of Example 1 and was found to have strong antitumor activity as shown in Table 7.

Table 7 112s1G ip 5 Li1 85 27
5:1 [101α39 0,40 ◆Bran 11! Using 5ill liquid, the amount of activated omvii* element precursor in turnip side crab hemocyte extract was measured in the same manner as in Example 1.

Table 8 As shown in Table $, polysaccharide 112S is 1G and 10
-” q/d shows strong coagulation enzyme precursor activation ability.9.

Reference Example 1 A thin OS solution such as p-su, dextofun, huminarin, and imelin, which is not known to have an antitumor effect, was tested using the O method in the same manner as in Example 1.
No 4 positivity was shown at any concentration between 0-7 and 10-' fll/dO.

Claims (1)

[Claims] Turnip F crab blood cell extract contains *purchased endotoxin V.
1. A method for screening polysaccharides having antitumor activity, which comprises contacting a diluted polysaccharide solution with a polysaccharide containing a polysaccharide, and selecting polysaccharides that show a positive Tsum W test.