JPH03205402A - Lignin glycoside and its use - Google Patents

Abstract

PURPOSE:To obtain a lignin glycoside which can satisfactorily be used as a medicine having anticancer action, wherein lignin and a polysaccharide are bonded in a specified bonding ratio, the polysaccharide is composed of uronic acid and a neutral saccharide, and the molecular weight lies in a specified range. CONSTITUTION:A lignin glycoside having the following properties: (i) lignin and a polysaccharide are bonded, (ii) the molecular weight is 60000-140000, (iii) the bonding ratio between the lignin and the polysaccharide is 1:1-20:1 (in terms of a molecular ratio), and (iv) the polysaccharide is composed of 60-70% uronic acid and 30-40% neutral saccharide. The lignin glycoside is prepared in the following way. Examples of the starting materials include tea, lithospermi radix, sophorae subprostratae radix, radix ginseng, pinecones and pine needles. The starting material is treated with a solvent such as hot water or ethanol and extracted with an alkaline solution. The pH of the extract is adjusted to 4-6. Ethanol is added to it, and the precipitated fraction is recovered. This fraction is purified by gel filtration, and the active portion is recovered. The lignin glycoside thus obtained may be subjected to dialysis, centrifugation, lyophilization, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industry] Second Field of Application] The present invention relates to a novel lignin glycoside and its uses.

[Prior Art/Problems to be Solved by the Invention] Most of the existing anticancer drugs have the effect of suppressing DNA synthesis or cell division, but they also have the same effect on normal cells. Cancer cells divide slightly faster than normal cells, which is a slower rate, so treatment is possible by giving more damage to cancer cells. Damage to normal cells + f is expressed as side effects, and the extent to which the body can tolerate these side effects is an important point in cancer treatment.

As mentioned above, original cancer treatment should be rooted in the biology and biochemistry of cancer cells, but in reality, {. It has not been linked to other cancer treatments.

Well, when it comes to the causes of cancer, three things have long been said to be carcinogens, radiation, and viruses. Among them, it became clear that the genetic information contained in cancer viruses causes cells to become cancerous, and oncogenes. The word (oncogene) was born. Later, it was hypothesized that oncogenes exist in normal cells, and that they are switched on at certain times, causing the cells to become cancerous.

This idea has evolved over time, and today everyone agrees that it was largely correct.

On the other hand, the genomes of higher animals contain pro-oncogenes.
There are more than 50 types of to-oncogenes, and they play important physiological functions in the proliferation and differentiation of normal cells. Therefore, the possibility of controlling cell proliferation and cancer at the gene level or gene product level has arisen. The purpose of the present invention is to develop a cancer therapeutic agent that suppresses the stage of cancer gene expression using a specific inhibitor. Using mouse mammary cancer cells in which the expression of the inserted mouse mammary tumor virus (ii) ITV) gene is regulated by corticoids, the MMTV gene expression is triggered by the depoly ADP-ribose reaction in chromatin proteins. has been found. That is, it is thought that as polyADP-ribose is degraded, local changes in the chromatin structure in that region ultimately lead to binding of RNA polymerase to the promoter and promotion of transcription. (Journal Biology Calke Mostory, 258:1537
1 (1983)].

Therefore, the inventor predicted that if the decomposition of polyADP-ribose was prevented, the oncogene would not be activated, so the inventors introduced poly(ADP-ribose) glycohydrolase, an enzyme involved in the decomposition of ADP-ribose, into humans. As a result of separating and purifying the placenta and examining compounds that have an inhibitory effect on this enzyme, we found that several natural compounds have strong inhibitory activity.

Also, this compound has llf! We discovered that tumor necrosis factor (TNF) has a completely new activity that enhances the cell-killing and cell differentiation-inducing effects, and that this enhancement effect is due to increasing the binding affinity of FNF to the TNF receptor. Furthermore, they discovered that this compound has the effect of inducing the production of TNF and interleukin l from macrophages.Then, they further investigated the effects of poly(ADP-rehose) glycohydrolase, The present invention has been completed by discovering a new compound that can be used as a drug having anticancer effects based on the induction of cytokines, including TNF, and their action-enhancing effects.

[Means to solve the problem]

That is, the present invention has the following gist.

■ Lignin glycosides with the following properties (i) Lignin and polysaccharides are bound (i]) Molecular weight is 60,000-140,000 (mountain) The binding ratio of lignin and multi-wMs is 1:1-201 (molecular ratio) (iv) Polysaccharide is 60-70% uronic acid, 1.30% neutral
~40%.

■ Poly(ADP--) whose main component is lignin glycoside
ribose) glycohydrolase inhibitor.

■ Cytolycoin, which is mainly composed of lignin glycosides, is a tumor necrosis factor (TNF) action enhancer.

■ Cytokine (TN) whose main component is lignin glycoside
F, IL-1) production inducer.

■ Cancer immunotherapeutic agents that contain lignin glycosides as the main component (in combination with cytotoxic agents such as TNF).

The lignin glycoside of the present invention comprises lignin and Ilt (polymer
It is a conjugate with l). Lignin and sugars (polyvMilQ)
The bond with is through an ether bond. The binding ratio is exemplified to be about 1:3 to 5 in terms of weight ratio of lignin to structural sugar. Further, the molecular ratio of lignin to polysaccharide is exemplified by about 1 to 20:1.

The sugar moiety of lignin glycosides is more sensitive than uronic acids and neutral sugars. Examples of its composition strength include 60 to 70% uronic acid and 30 to 40% neutral #M.

Examples of neutral sugars include glucose, galactose, mannose, and arabinose. The main ingredients are glucose 15~201101% and galactose 25~201101%.
Examples include 30 mol %, mannose 35 to 50 mol %, and arabinose 10=15 mol %.

These constituent sugars as a whole are I! It has a chain structure,
Forms polysaccharides.

The molecular weight of the lignin glycoside is about soooo to 140,000, and the molecular weight of the polysaccharide part is about 40,000 to 100,000. In addition, the lignin part has a molecular ffi of 1000 to 1000.
It has about 0.

Elementally, the lignin glycoside of the present invention has 35 to 45 C atoms.
Examples include 1 to 10% by weight of H atoms, and 45 to 64% by weight of 0 atoms.

The lignin glycoside of the present invention is prepared as follows.

Examples of starting materials include tea (leaves/branches), purple root, three bean root, Korean ginseng, pine (cap/leaf), and grass roots (trunk).

The raw materials are mixed with various solvents (e.g., hot water, ethanol, acetic acid, etc.).
processing). The processing time is about 1 to 15 hours. Extract the treated raw material with an alkaline solution (0.1-IN sodium hydroxide, ammonium, etc.). The extract is p
Adjust to H4-6, add 15 times the volume of ethanol, and collect the precipitate fraction. The precipitated fraction is purified by gel filtration to recover the active portion.

The lignin glycoside thus obtained can be subjected to dialysis, centrifugation, freeze drying, etc.

The lignin glycoside of the present invention is poly(ADP-ribose)
It has a glycohydrolase inhibitory effect, and an effect of inducing TNF production and enhancing its effect, and is effective in mammals including humans (
humans, horses, dogs, mice, mormons, rats, etc.)
It has poly (ADP-ribose) glycohydrolase inhibitory activity against poly(ADP-ribose) glycohydrolase and activity of inducing the production of PNF and enhancing its action.
The compound is useful for the treatment and prevention of malignant tumors and viral infections as a TNFase inhibitor and an activator for inducing TNF production and enhancing its action.

The lignin glycosides of the present invention are administered orally or parenterally.

The lignin glycosides are administered in the form of a pharmaceutical formulation, either by itself or with a pharmaceutically acceptable carrier.

The formulation is prepared by methods known per se.

Examples of dosage forms include tablets, capsules, powders, suppositories, and injections.

For example, when administered orally, lignin glycosides usually have a concentration of 0.
1 ~]. O O mg/kg body weight] IE
I I ffj+ or administered over several times,
The dosage may vary depending on age, weight, and/or the severity of the condition being treated and response to treatment.

Toxicity Experiment Regarding the toxicity of the lignin glycosides of the present invention to mice, the LD5o value was 100 μ/kg or more when administered orally, and the LD5o value was lower than the administered dose. It is a compound with an extremely high value and a wide safety margin.

[Example] Example 1 Lignin glycosides were extracted by the following process.

Example pine cone ↓ leaving water boiling time and amount of pine cone?
■8 Pine cones extracted with ethanol? ■Yu■ Add an equal amount of ethanol to the extract (I) Freeze-dry the solution after dialysis Example 2 - Sugar moiety of gunin glycoside, glycone
In order to investigate the structural characteristics of the non-sugar moiety and aglycone, this glycoside was subjected to methanolysis (methanol-hydrochloric acid decomposition) or chlorite (NaCl).
The glycone and aglycone were separated and analyzed using the O■) method. The results are as follows.

[Glycon analysis]

Molecule I: Sepharose CL-4
B Gel filtration method: 60,000 to iooooo Sugar composition 1 (%) (Total weight %) Uronic acid 63.4 48.8 Neutral sugar
36,6 28.2 It has the characteristic that two-thirds of the glycon is uronic acid.

Neutral sugar composition (mol %) glucose
182 Galactose 27.4 Mannose 40.2 Arabinose 13.8 Fucose O Neutral sugars include glucose, galactose, mannose, and arabinose, but do not include fucose.

a) Uronic acid was determined by the carbazole method, and neutral sugars were determined by the phenol-sulfuric acid method.

b) Neutral sugar composition or methyl glycoside produced by methanolysis was trimethylated and analyzed by gas chromatography.

[Analysis of aglycon]

Molecular weight: Sepharose C
4000±2000 by L-6B gel filtration method Infrared absorption analysis (IR) IR was measured using a KBr disk.

As a result, 3400 in the 3500-3700 cm-' range
Absorption with a peak at crV' was detected. This absorption indicates the presence of phenolic hydroxyl groups. Absorption near 1600 cm indicates an aromatic double bond. The absence of carbonyl group absorption near 1700 cm indicates that the compound does not have ester bonds as seen in tannins, but is polymerized with ether bonds as seen in lignin.

The entire subek I. rum, including the fingerprint region, is very similar to lignin (alkali).

From the above IR spectrum results, it is concluded that the aglycone of this glycoside is not a tannin-like compound but a lignin-like compound.

Ultraviolet absorption (UV) analysis: Maximum absorption value at 280 nm and minimum absorption value at 260 nm.

280/260=1.02 Lignin also has a similar UV spectrum of 280/26
0=1.03 UV spectrum indicates the presence of aromatics (probably phenols).

Electron spin resonance (ESR) analysis shows that a g=2.004 EsR signal, similar to that of lignin, is detected, indicating that the structure contains stable free radicals. Note that no such signal is observed for tannins.

(Analysis of lignin glycosides) Characteristic elemental analysis of the entire lignin glycosides (weight%) C 3 9. 8 3 H 4.41 0 5 5. 7 3 N 0. 0 3 or less S O Nitrogen, Since it does not contain sulfur, it does not contain proteins or sulfate groups.Sepharose CI-4
The molecular weight determined by the B gel filtration method is approximately 110,000.

The binding ratio of lignin and sugar is approximately 1=4 by weight.

Therefore, this glycoside has the characteristic that it contains about 50% of the total weight of uronic acid as a sugar moiety (glycone). Neutral sugars include glucose, galactose, mannose, and arabinose.

The non-sugar moiety (aglycone) has certain characteristics from lignin.

This glycoside is a ○-glycoside in which the reducing end lactol hydroxyl group of sugar is dehydrated and condensed with the alcoholic or phenolic hydroxyl group of lignin to form an ether-like bond. In addition, the binding ratio of lignin and sugar is approximately 1:4 by weight, and in particular, it is a lignin glycoside (when classified by aglycone characteristics) with a molecular weight of approximately 110,000 liters that contains a large amount of uronic acid, which is an acidic sugar. be.

The estimated structural model of xylem glycosides is as shown in the drawing.

Test example l. Hasofafor (0.01% bovine serum albumin-10mM mercap I ethanol-50mM potassium phosphate, pH 7.0) for assaying the inhibitory effect on poly(ADP-ribose)glycohyperolase, H-(AD
P-ribose) n+I5 was added, and to the 27 μC was added poly(
ADP-ribose) glycohydrolazene solution was added to bring the total volume to 30 .mu.C, followed by incubation at 37.degree. C. for 1 hour. After that, the reaction solution was absorbed into DE81 filter paper, and the filter paper was washed with water, ethanol, and acetic acid.
It was dried, and the unreacted group t3H-(AD
P--ribose) was measured to examine the inhibitory effect of the test substance on this enzyme. Table 1 shows the results, and all of the test substances used showed poly(ADP) in a dose-dependent manner.
-ribose) glycohydrolase.

Table 1 Poly(ADP-ribose) glucohydrolase inhibitory activity of lignin glycoside Test Example 2 Inhibitory effect on gene expression The gene expression system used in this test was
Mouse mammary tumor virus (MMTV), a susceptibility gene
The 341 mouse breast cancer cell line containing the gene was used. In the presence of glucocorticoids, this cell
SRNA and further spliced 24SRN
It expresses two types of A. This expression is based on the cDNA of the env part.
It can be detected by using A. Therefore,
The test substance was added to 341 strains at a concentration of 30 μg/form, and 3
After incubation at 7°C for 30 minutes, dexamethasone was added to the system at 10-'M and incubation was continued for an additional hour. After that, the 3IlI cells were collected, and high molecular RNA was extracted using the guanidine-hydrochloric acid method.
Treated at 60°C for 5 minutes (20 mM Ml) PS,
pll7.0.5mM sodium acetate, 1mM
DTA), then 1.2% agarose gel (vanofar)
) Electrophoresis (40V, 16h) was performed using a C trowel. Then, 1 transfer to nitrocellulose, 32 [)
The DNA-TV11N^ (env-specific cDN8) was hybridized and autoradiodram was generated using X-ray film. Thereafter, the concentration of 35S and 24S RNA bands from the autoradiodrum was measured using a densitometer to measure the amount of RNA expression, and the RNA expression suppression ratio was calculated by comparing with the case without the addition of the test substance. Table 2 shows the results, and the test substance used showed an effect of suppressing MMTV gene expression.

Table 2 Effect of lignin glycosides on breast cancer virus expression (MMTV) gene Dexamethasone Lignin glycoside C10-7M). C30ug/ml) 353
2 4S band photosensitivity + + 10+ ++ 10+ 10++ l9 l is positive control, 2.3 is negative control, 4 is experimental group test example 3
．． Mouse Experiment III Anticancer Effect on Tumors Sarcoma 180 tumor cells were injected into the peritoneal cavity of mice at 1×1.
06 cells were transplanted, and the test substance was continuously administered intraperitoneally for 1 to 4 days after transplantation. Antitumor activity was determined from the survival rate compared with the physiological saline administration group.

The experiment was terminated on the 45th day after tumor implantation. Table 3 shows the results, and the test substance used showed anticancer activity.

Table 3 Mouse tumor Sarcoma 180! Anticancer effect of sullignin glycoside Lignin glycoside 4 0×4 20×4 1 0×4 5×4 1 0 8 2 0 6 120 91 Lignin glycoside was administered for 4 consecutive days from the day after transplantation. administered.

Test example 4. Enhancement effect of TNF action on mouse fibroblastoma cells L-929 The cell-killing action of TNF utilizes the adhesion of L-929 cells to petri dishes, and the surviving living cells are stained and the staining concentration is measured. It can be measured by Therefore, the test substance is added to L-929 cells, then actinomycin D is added at a concentration of 4 μg/d, TNF is further added, and the cells are incubated at 37'C for 18 hours. The plate is then washed with saline to remove dead cells.

Thereafter, the living cells were stained with 0.1% crystal violet, the stained living cells were lysed with 0.5% SDS, and the dye concentration was measured using ○D590ni.
The enhancement effect on F action was investigated.

Table 4 shows the results, and all of the test substances used enhanced the cell killing effect of TNF in a dose-dependent manner.

Table 4 TNF effect on mouse fibroblast L929 Enhancement effect Lignin glycoside concentration (μg/1) CD of TNF. Concentration Cg/m branch) Enhancement magnification (%) 0 0. 0 3 4
1 0 03 0.015
2211 0 0.
0 0 6 2 5 4 B3 0
0. 0 0 4 4 7 7
3100' 0.0074
458 Test Example 5 Anticancer effect of combined use of TNF against mouse experimental tumor Six Sarcoma 180 tumor cells were subcutaneously transplanted into mice, and the test substance and TNF were continuously administered for 1 to 5 days after the transplant. Antitumor activity was determined from the survival rate compared with the physiological saline administration group.

The experiment was terminated on the 45th day after tumor implantation. Table 5 shows the results, and the test substance used was T”N F
It showed strong anticancer effect when used in combination with

Table 5 Mouse II! Effect of combined use of lignin glycosides and TNF on II tumor Sarcoma 180 Sample Concentration T/C (■/kg
) (%) 0 'T' N F "FNF + Lignin glycoside 100 118 40X4 191 20X4 298 10X4 254 5x4 188 Lignin glycoside was administered together with TNF every day for 4 days from the day after the transplantation day.

[Brief explanation of drawings]

The drawings show the quasi-defined structure of the lignin aphrodisiac of the present invention. 23 1: sugar 2: lignin 24

Claims (5)

[Claims] (1) A lignin glycoside having the following properties. (i) Lignin and polysaccharides are combined (ii) Molecular weight is 60,000 to 140,000 (iii)
The binding ratio between lignin and polysaccharide is 1:1 to 20:1 (molecular ratio) (iv) Polysaccharide has 60 to 70% uronic acid and 30 to 30% neutral sugar.
It is made up of 40%. (2) A poly(ADP-ribose) glycohydrolase inhibitor containing lignin glycoside as a main component. (3) An enhancer of cytokine action whose main component is lignin glycoside. (4) An inducer of cytokine production whose main component is lignin glycoside. (5) Cancer immunotherapy agent containing lignin glycoside as a main component.