JPS5940136B2 - Antitumor agent consisting of purified lignin sulfonic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to lignin sulfonic acid (hereinafter simply referred to as L) obtained from the steaming effluent (hereinafter simply referred to as SSL) discharged when wood is steamed into pulp by the sulfide method.
The present invention relates to an antitumor agent that is purified LSA consisting of LSA (abbreviated as SA) and from which low molecular weight moieties have been removed.

As mentioned above, SSL' refers to the steaming effluent when wood is steamed using the sulfide method to obtain pulp. For example, the composition of hardwood SSL anhydrous is 40 to 45% LSA (hereinafter referred to as
3 to 5% of hexose, 15 to 20% of pentose, 15 to 20% of sugar modified product, 10 to 15% of organic acid furfural, and 5 to 10% of inorganic material.

The raw material LSA used in the present invention is SSL isolated by dialysis using a cellophane membrane or cellulose acetate membrane, reverse osmosis, or ultrafiltration method, and the purity of this LSA is about 70% or more. be.

Even in the case of the above-mentioned raw material LSA, it may have excellent antitumor properties with low toxicity, but it has the drawback of large variations in its properties. The present invention was completed as a result of various studies aimed at obtaining purified LSA (abbreviated as purified LSA).

The raw material LSA from which the low molecular weight segment with a molecular weight of less than 400 has been removed has stable quality as an antitumor substance.
It was found to be less toxic.

To obtain purified LSA, raw LSA is usually subjected to gel filtration, but the dialysis method, reverse osmosis method, etc. used to obtain raw LSA may be repeated.

In general, the molecular weight of L'SA ranges from 200 to several hundred thousand, and L'SA
SA is a water-soluble polymer with a complex structure.

Purified LSA is obtained by removing the portion with a molecular weight of over 10,000 from raw LSA using a cellophane membrane, etc., and then removing the portion with a molecular weight of less than 400 by means such as gel filtration (hereinafter abbreviated as purified dialyzable LSA). It has the most stable quality, low toxicity, and excellent antitumor properties.

Among purified dialyzable LSAs, the molecular weight is 400-2000.
is the best in terms of quality.

Furthermore, LSA made from hardwood is superior to LSA made from softwood in terms of antitumor properties.

Previously, one of the present inventors has found that lignin, SSL, and LSA in food ingredients are found in mice with implanted tumor sarcoma 180.
Noting that LSA exhibits an inhibitory effect on
It was found that the antitumor ability of LSA was changed by the chemical treatment, but it was not yet clear by what mechanism of action the antitumor ability of LSA was changed by these chemical treatments.

However, in order to clarify the antitumor performance of LSA without chemical treatment, we conducted a detailed study of the fractionated fractions of LSA and discovered that toxic substances were mixed in the raw LSA in the molecular weight category of less than 400. However, the present invention was achieved by discovering that the antitumor properties of LSA can be stabilized and the toxicity can be reduced by selectively removing them.

The dialysis fluid (hereinafter referred to as
The crude dialysable LSA is further divided into low molecular weight fractions of less than 400 by Sephadex G-500 column chromatography. Purified dialysable LSA was obtained from which .

Note that there is no low molecular weight category of less than 400 in non-dialyzable LSA.

The ultraviolet-visible absorption spectra of this non-dialyzable LSA and purified dialysable LS'A were measured in alkaline solution and acidic solution, and the difference was obtained by subtracting the absorbance value of the acidic solution from the absorbance of the alkaline solution at each wavelength. The spectrum (abbreviated as Δεi spectrum) is shown in the figure.

The measurement was carried out by measuring the UV-visible absorption spectra of both LSA aqueous solutions adjusted to pH 12 with NaOH and pH 6 with phosphate buffer, and calculating the absorbance value of the acidic solution from the absorbance value of the alkaline solution at each wavelength. I subtracted it.

The Δεi spectra of both LSAs showed a peak derived from free phenol at around 300 nm and a peak derived from phenol conjugated with an α-carbonyl group at around 350 nm.
It shows absorption of A%.

However, there is a difference in the absorption intensity of both LSAs, and the results of measuring each phenol group using the method of Goldschmidt et al. show that the free phenol hydroxyl group of non-dialyzable LSA is 21
6%, and the amount of phenolic hydroxyl groups conjugated with the α-carbonyl group was 0.12%.

On the other hand, the amount of free phenolic hydroxyl groups in purified dialysable LSA is 4.98%, which is more than twice that in non-dialysable LSA, and the amount of conjugated phenolic hydroxyl groups is 0.98%, which is more than three times that in non-dialysable LSA. It was 39%.

The free phenol content in LSA and antitumor properties are not necessarily parallel, and LSA with high antitumor properties tends to have a high free phenol content, but the present inventors have found that the opposite is not always true. This can be said from the results of their experiment with periodic acid oxidation.

This suggests that the amount of free phenol in LSA is not a functional group directly involved in the expression of antitumor activity, but is rather an auxiliary factor that enhances the efficacy of other functional groups.

Although the nature of the functional group involved in the expression of LSA's antitumor ability is not clear, the antitumor ability of purified dialyzable LSA is higher than that of purified LSA, and the structure having a phenolic hydroxyl group conjugated with an α-carbonyl group may be associated with tumorigenicity. It is presumed that antitumor ability is generated by reacting with cells.

It can be said that the free phenol or sulfone group coexisting in LSA acts as a cofactor that contributes to changes in the redox potential of cells and increases in hydrophilicity.

There are three ways to administer purified LSA as an antitumor substance: oral administration and intravenous injection.

In the case of oral administration, the dose is administered as a tablet at 300 to 2,000 μg/kg body weight, approximately 182 times, or 50 times in total.

In the case of intravenous injection, an amount equivalent to 50 to 500 μg/kg body weight per dose is dissolved in Ringer's solution and administered approximately 3 times a week, a total of 2 times.
0 times.

Therefore, the effective dose is 15~ for oral administration], OO
? /kg body weight, 1-10 g/kg body weight for intravenous injection.

The acute toxicity of purified LSA was determined by Food and Cosmetics Toxicology (Fd, Cosmet).

As a result of testing and testing according to the method described in Toxicol, ) Vol. 11.229 (1973), LD50 = 4 OL? in mice (oral). / ”
I weight and ivy.

Next, the effects of the antitumor substance according to the present invention will be explained using specific examples.

Example Hardwood 5SL (solid content 15.3%) 11 was dialyzed in tap water with a cellophane membrane for 5 days to remove reducing sugars and sugar-modified substances, and the dialyzed solution was concentrated to dryness under reduced pressure at 50° C. or lower. Thus, 35 g of raw material LSA powder was obtained.

Dissolve 0.57 of this raw material LSA powder in 20 ml of deionized water, and
rL) and developed with deionized water.

While measuring the absorbance of the effluent at 400 nm, 200 ml of the polymer fraction corresponding to a molecular weight of 400 or more was collected as a fraction of the chromatogram, and purified L was lyophilized.
0.41' of SA powder was obtained.

In addition, 10'iI of raw material LSA powder was dissolved in distilled water and 1
0% solution, and this solution was again placed in a cellophane membrane at 201
Dialysis was performed for 24 hours against distilled water.

When the dialysis fluid was replaced and this operation was repeated, the dialysis fluid hardly showed any absorbance at 280 nm.

At this point, dialysis is completed, and the dialysis fluid no longer contains dialysable LSA.
dialysis fluid (non-dialysable LSA).
) was discarded.

The external dialysis solution was concentrated to approximately 300 ml under reduced pressure at 50°C or below, and then freeze-dried to obtain a crude dialysable LSA powder of 4.62 ml.
I got it.

0.59 of crude dialyzable LSA powder was dissolved in 20 ml of deionized water, Sephadex G-50 (3.5 x 110 cI
TL) column to obtain purified dialyzable LSA powder 0.4:l' having a molecular weight of 400 or more.

A comparative test of the antitumor effects of the four types of raw LSA, purified LSA, crude dialysable LSA, and purified dialysable LSA was conducted using 10 mice in each test group.

0.2 ml of 105 ascites-type sarcoma 180 cells were intraperitoneally transplanted into the peritoneal cavity of a 4-week-old ddN detached mouse weighing 207 cm, and 24 hours later, the mice were intraperitoneally injected five times every other day for 10 days.

The reason why male mice were used in this study is that female mice sometimes develop spontaneous breast cancer, which may complicate analysis of the results.

One dose depends on the abundance ratio of LSA, raw material LSA,
Purified LSA, crude dialysable LSA, and purified dialysable LSA were set at 50.23.47.20 m9/kg body weight.

Mice in the non-administration group were only implanted with tumor cells.

This is because it was known that intraperitoneal injection of only 0.2 rnl of distilled water or Ringer's solution did not significantly affect the growth of tumor cells.

After the above cell transplantation, the survival rate was compared on days 15, 19, 23, 27, and 29, the body weight was measured, and an autopsy was performed on all mice immediately after death or at the end of the test to determine the presence or absence of ascites retention. Changes in internal organs were observed.

The results are shown in the table below.

As is clear from the table above, purified LSA, purified dialyzable LSA
In the group, 70.90% of the mice in the non-administered group died even on the 27th day, showing excellent antitumor effects.

After the test was completed, an autopsy was performed on the nine surviving mice to which the purified dialysable LSA of the present invention had been administered, and as a result, no accumulation of ascites was observed in any of them.

The fact that ascites accumulation due to Sarcoma 180 cells could not be detected at this point suggests that Sarcoma 180 tumor cells will not begin to proliferate again in the bodies of these surviving mice, and these individuals are considered to have been completely cured. It was judged.

On the other hand, all the mice in the LSA group, which is the raw material for the control product, died around the 20th day, which was earlier than in the non-administered group.

The mice in the control crude dialysis LSA group followed almost the same course as in the non-administered group, and all mice died.

However, the weight change of the mice in the crudely dialyzed LSA group was different from that in the non-administered group, and the degree of weight increase was relatively slow, indicating that ascites accumulation was not significant.

Therefore, it seems likely that the mice in this area died mainly due to the proliferation of tumor cells (although it is thought that the toxic substance may have had an effect).

In contrast, mice in the untreated group weakened and died due to the proliferation of tumor cells.

In addition, no weight loss or other side effects were observed in mice treated with the purified LSA group or purified dialysable LSA group of the product of the present invention, proving that it is extremely effective compared to conventionally used antitumor agents that have strong side effects. Ta.

[Brief explanation of drawings]

The figure shows the Δεi spectra of non-dialyzable LSA and purified dialysable LSA. In the figure, solid line: purified dialysable LSA, dotted line: non-dialysable LSA
0

Claims (1)

[Scope of Claims] 1 Raw material lignin sulfonic acid isolated from wood sulfide valve drainage by dialysis, reverse osmosis, or ultraviolet filtration is subjected to gel filtration treatment to remove low molecular weight fractions with a molecular weight of less than 400. An antitumor agent consisting of purified lignin sulfonic acid. 2 The molecular weight of purified lignin sulfonic acid is 400 to 100
00. An antitumor agent comprising purified ligninsulfonic acid according to claim 1.