JPS60130525A - High polymer immunosuppressant obtained from adlay and production thereof - Google Patents

Abstract

NEW MATERIAL:An immunosuppressant CLJ-100 having the following properties; Elementary analysis; C, 39.5-41.0%; H, 5.8-6.0%; N, <=2.5%; ash, 0%. Molecular weight; 100,000. Melting point; Showing no definite melting point, but carbonizing at >=200 deg.C. Specific rotatorypower [alpha]<25>D+126.5 deg. (C=0.1%, 0.01N-NaOH aqueous solution). Solubility; Soluble in water and insoluble in organic solvents. Color reactions; Negative to iodine reaction. White amorphous substance. Sugar content; 80-90% as glucose and 8-16% uronic acid. Constituent sugar; Mainly containing detected glucose, galactose and mannose, etc. without detection of uronic acid nor amino sugars. USE:A high polymer immunosuppressant. PREPARATION:Seeds of a plant belonging to the genus Coix of the family Gramineae are extracted with hot water and a component having 10,000-300,000 molecular weight is fractionated from the resultant extract by the ultrafiltration method. The aimed immunosuppressant CLJ-100 is then separated and purified from the above mentioned component.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a molecular immunosuppressant obtained from Coix seed and a method for producing the same.

Coix barley has been used as a medicinal plant since ancient times in China and Japan, and herbal medicine containing it has been used to treat skin diseases,
It has not been applied to symptoms such as arm rheumatism. Previous research on medicinal ingredients has reported the presence of Colxenolide, which has antitumor activity, in the acetone-soluble part of seeds (T, Ukita et al.; Ch.
Ern Pharm, Bull, 9, 43-47
(1961) ) Also, 1-Colxiol present in the roots is said to have blood pressure lowering and analgesic effects [Koyama, et al.; Pharmaceutical Journal.

75, 702 (1955), 76, 100
2 (1956)).

However, these Co1xenolide and Co
The medicinal effects of 1xiol are not directly related to the medicinal effects of the above Chinese prescription.

Therefore, the present inventor focused on the fact that the medicinal effects of Chinese herbal formulas containing red bean seeds are closely related to allergic diseases, and as a result of examining the immunosuppressive effects of the herbal medicines that make up the formula, we found that We confirmed in animal experiments that there is a component that significantly inhibits the production of IgE, which is the main cause of allergies, and as a result of our efforts to reduce the amount of active component*F#H.
A polymeric immunosuppressant agent having the physicochemical properties described below was discovered, and this substance was named immunosuppressant CLJ-100.

The present invention was completed based on the above knowledge, and
Physical and chemical properties mentioned later? Immunosuppressants with and Poaceae-
・Seeds of plants belonging to the genus Atomyx are extracted with hot water, and components with a molecular weight of approximately 10,000 to 300,000 are separated from the extract by ultrafiltration (5), and the immunosuppressant C'LJ-100 is extracted from the components. Immunosuppressant CLJ-100 that can be isolated and purified
This is the manufacturing method.

The immunosuppressant CLJ-100 of the present invention has the following physical and chemical properties.

■Elemental analysis; C39.5-41.0%, 5.8-6.0
H, N2.5 H or less, ash content O%, ■Molecular weight: Approximately 100,000 (by gel filtration method), ■Melting point;
It does not have a clear melting point and carbonizes at temperatures above 200°C.

■Specific optical rotation; [α]”+126.5° (C=0.1°, 0OIN-NaOH aqueous solution, ■Ultraviolet absorption spectrum;
3. Infrared absorption spectrum; as shown in Figure 2, showing maximum absorption near 8 nm and 270 nm.
At around 300-1, 2940-'', and 1150--, there is preload and absorption that seems to be related to the depiction.

■ Solubility in solvents; soluble in water, non-bathable in organic solvents, Q) Color reaction; iodine reaction, 1 Korean, ■ Color and shape of substance: white amorphous, OD shield, sensitive; glucose 80-90% (by phenol #L acid method), 8-1 uronic acid, 6% (by carbazole sulfuric acid method) [Co.] 41 phosphoric acid; according to trifluoroacetic acid hydrolysis, mainly glucose is synthesized, Galactose, mannose or arabinose, and galactutetraphosphoric acid are detected, but uronic acid and amino sugars are not detected.

In addition, the present immune suppressant CLJ-100 has the following biochemical properties.

■ Acute toxicity: 2500 m/KyC mouse, intraperitoneal injection ■ The present invention suppresses IgE production to less than about 2 layers by 2,4-dinitrophenylated chicken egg albumin antigen (rat PCA reaction). It is a sugar polyshield-like substance of the immunosuppressant CLJ-10Old'. Therefore, the above Co1xenolide +Co1x
Enol is completely different. Glycyrrhizin [Takeo Miyake: Allergy 10゜131 (1961) and others] is known as a substance λ obtained from plants that has a suppressive effect on elephant disease, but this is a low-molecular substance with a molecular weight of 823, and it is different from this substance. Obviously # (J4. Also, licorice, psycho,
It has been reported that pigeon molecules also have an immunosuppressive effect on monkeys and other species [Matsu Nakashima et al.: Record of the 3rd Keihanna Symposium 98° (1969), S. Nak.
Ashima et al.: Proc.

Symp, WAKAN-YAKU 13, 42 (
(1980) ), but their Kt-1 immunosuppressive activity is also much lower than that of the substances of the present invention, which differ from the plant of origin. In addition, the peptidogen V-can found in tuberculosis bacteria has an immunosuppressive effect [Tenji Nakanishi et al.: Japanese Society of Immunology General Meeting Record 8, 7
(1978)], with different chemical properties. Therefore,
・・Immunosuppressant CLJ-1 made from isolated red bean sprouts
00 is a previously unknown inclusion substance.

Immunosuppressants are generally used for the prevention and treatment of allergies, autoimmune diseases, and other illnesses.
3.J) - Inhibits all specific activity and plays an important role in preventing infection, such as IgG and IgM.

The aill renal steroid hormone cirrhizin, which is widely used as an anti-allergy agent, suppresses the production of all immunoglobulins, so long-term use can cause side effects and increase the risk of infection with other diseases.
-100 is chemically structurally completely different from adrenal steroids, etc., and it significantly suppresses the production of IgE, which is thought to be the cause of type I allergy, according to Kamaki's animal experiments.
Since it has little suppressive effect on other immunoglobulins such as gM, it is useful as an immunosuppressant having preventive and therapeutic effects on allergic diseases thought to be caused by IgE.

Examples of plants used in the present invention that can be deceived by the stripes of Colay of the Poaceae family include Coix of the Grass family, which is grown in Southeast Asia, China, Japan, and the like. The seeds from which the pericarp and skin graft have been removed are called titan seeds.

If the pericarp and skin grafts are not removed, it is called huanggi with skin or adlay. In addition to the above, there is also the wild type Juzudama that grows naturally in various parts of Japan. This seed is called husk in the market.

...The extraction efficiency of the active ingredients is extremely low if the tomato is not crushed, and the surface of the tomato husks is particularly strong and it is difficult to crush, so for practical purposes, refined yitsutane is used as the raw material. It is commonly used as a tomato, but the price is higher than that of otomugi, so you can also use it by crushing otomugi.

In order to increase the extraction efficiency of the present immunosuppressant CLJ-100, the above-mentioned plants (11!) are first crushed. When extracting the present immunosuppressant CLJ-100 from the various crushed plants with hot water is preferably treated with an organic solvent such as acetone in advance.

This treatment operation is carried out by immersion in an organic solvent for several days or by heat treatment at 60 to 80°C for 1 to 2 hours. By this organic solvent treatment, the above-mentioned Co1xeno
Lide and other oil and fat components can also be removed at the same time.

Hot water extraction is performed using water in an amount several to ten times larger than that of the raw material. Extraction can be carried out at a temperature of 50 to 120 degrees Celsius (1W), but extraction efficiency is low below 60 degrees Celsius, and at high temperatures above 90 degrees Celsius, gelatinization of starch may cause contamination in the subsequent separation and purification process. Therefore, it is preferable to extract at a temperature range of about 60 to 85°C. The extraction time is usually 2 to 4 hours, and the water may be replaced 2 to 3 times for 1 to 2 hours each.

The extract obtained in this way contains many substances such as color stains, low-molecular substances, salts, and starch in addition to the present immunosuppressant, so these impurities are removed and the active ingredients are condensed. Ultrafiltration is performed to Ultrafiltration can be any method that efficiently collects polymer fractions, but at least molecules! If you use ultrafiltration to separate out the 10,000 to 30,000 components of soil, you should do it.

Examples of the ultrafiltration device t include Dia 70-Cell 8200 type and 8400 type manufactured by Amicon in Japan, Ultrafilter UE (P-76 type and UHP-62 type manufactured by Toyo Kagaku Sangyo Co., Ltd.), and Vericon Lab manufactured by Millipore in the United States A cassette or the like is used.

Examples of ultrafilter membranes include Amicon's XM300 type, Xy1100A type, YMIO type, and 71%42 type, and Kayo Kagaku Kagyo Co., Ltd.'s Ultra Filter 0K200. IJK5
0, UKIOlOKl, etc. These ultrap filters equipped with ultra p filter membranes are used in combination. @
For example, an extract is subjected to limit σ1 with The VI liquid is separated into the fJi liquid with a YMIO membrane, and the components with a molecular weight of 10,000 to 300,000 that do not pass through this are fractionated and the molecules that pass through the filter are less than &rquo;10,000. The components are separated into a filtrate.

In this way, the extract was fractionated by the ultra-r groove method and PC
Separation of all known components from 10,000 to 300,000 molecules! The present immunosuppressant CLJ-100 may be separated and placed in a blue J column using the t1 production method.

Bunliao Jinghui uses gel filtration method, ion exchange chromatography method,
It is carried out by combining various methods such as chromatography and chromatography.

As a gel FJM agent, if all commercially available gel filtration agents with suitable fractionation ranges are used, components with a molecular strength of 10,000 to 300,000 can be obtained by one technique under ultraviolet light and the phenol sulfurization method (611 determined from It can be divided into θ fractions from 1iJ-11.For example, agarose-based gel PffM such as Bio-Rad's Biokel A1 and Pharmacia's Sepharose CL6B.
By counterfeiting the entire active ingredient, the active ingredient can be completely separated.

When performing ion exchange chromatography, 14 ion exchangers such as Pharmacia's C↓ Tetsu Sepharose and Toyo Rudatsusha's mcM Toyovar pass through without being adsorbed, but the negative Ion exchanger, e.g. DEAE-Sepharose manufactured by Pharmacia, Bioken Rad HD
The active ingredients are adsorbed at a moderate pH in EkE]kuyogel, Toyo Wtasha MDEAE-Toyonokuru, etc.
Subsequently, when salt is added to the elution buffer, the salt is eluted within a certain concentration range.

The amount of sugar measured by either the gel E filtration method or the ion exchange chromatography method is usually 275 nm ultraviolet acid absorption and/or phenol (sugar measured by the PLT acid method). , it can be fractionated into several fractions depending on its value.

The extracts of the same fraction were mixed, salts and buffer components were removed by Renhui's method such as vacuum filtration and dialysis, and the desired immunosuppressant CLJ-100 was obtained by freeze-drying. It will be done. Usually, the above-mentioned Kel filter agent is eluted first, and the anion exchanger is eluted first, and after being soaked in water, it is washed out with a buffer solution containing 0.2M sodium chloride.

Obtained in this manner/Slack Throwing Inhibitor CLJ-1 (10
In the claimed state, it is a stable white amorphous substance and is considered to be juvenile.

Next, a method for measuring the IgE suppressing activity of the present immunosuppressant CLJ-100 will be described.

l) Inhibitor and antigen administration method for 1gg production
．． 0 Ilp were collected, each dissolved in 2.0 ml of water and 5 ml of Q per mouse was administered into the r+> cavity of 3 groups of mice (3 mice per group) at 6 days of age. J1 Il
1lill 8+ M' gives 1 cancer to group 1.0η, 8 groups 0, 5 mg, 0 group 0925 l. On the day of administration and after 1-12 hours, administer 10 μg of 2,4-dinitrophenyl ovalbumin per mouse to 4 liters of alum duband.
σ) Administer intraperitoneally along with the suspension. As a control, the same antigen was administered together with an adjuvant to the same group of 3 mice that had not been administered the sample. 7. After 14 days of antigen administration, blood was collected from the mice by cardiac puncture, the blood from the 3 mice was mixed,
Separate the serum and dilute it 10x, 30x, 1x with fresh 1 + Ii co-saline solution.
00 times diluted serum.

2) Measurement of IgE by PCA method The hair on the back of a male rat before and after body weight t so, y was completely shaved, and ironically, the diluted blood of the above-mentioned labor mouse ('ff Q, l I
n1f, (Inject. 204 subsites can be administered per animal. Injection 3-4 hours tli-, dinitrophenyl egg albumin antigen ■0ηt 1% Evans blue solution 10ml 1K solution dissolved in rats) 1ml per animal Inject into the tail vein.

30 minutes after injection, kill the rat with chloroform anesthesia, peel off the skin on the back, and measure the heat dissipation and thickness of the rat. If the effect of the inhibitor is sufficient and there are no IgE antibodies in the blood, 6-color spots will be observed in each dilution of the serum, but if the inhibitor is weak or the inhibitor is not used. Control mouse blood r? In case J) 5, a marked evening-colored patch with a diameter of about 5 to 20 mm appears at the serum injection site. W
4 points for cases where the inclusion does not appear at all, and 4 points when the inclusion does not appear at all, and the inclusion appears as a partial arc around the injection site.

3 pieces with a diameter of 10 mm or less, 2 pieces with a diameter of 1
5mm @ (1 point for l, 0 points for diameter 20mm or more f: 0 points are given, and the scores for the same serum are totaled to judge. There was no visible embedding at all in each serum dilution, and the lifting platform received 12 points. Therefore, if all dilutions show d-rapidity of 20 mm or more, 0 points will be given.A total score of 9 points or more means that eggs are significantly suppressed, and 8 to
A score of 4 is evaluated as moderate inhibition, and a score of 3 or less is evaluated as almost no inhibition.

The present invention will be explained in detail with reference to an actual example.
This is merely an example and is not intended to be limiting.

Example I 500g of commercially available refined pearl barley (Jin Kuratori) was placed in a 34-valley Erlenmeyer flask, and 1.57 g of water was added. was added, the gold side of the reflux condenser was placed, and the mixture was heated for 3 hours in a water bath with a bath temperature of 85°C. The flask was cooled under running water, most of the liquid was removed by decanting, 500 ml of water was added to the residue and mixed well, and the p liquid was separated again by decanting. This was repeated two more times with the same water. All ingredients collected, 7000 r-p, -m at 30
Centrifuge for 1 minute and remove 1.71 minutes of supernatant. This was applied to an ultrafilter UHP-76 model manufactured by Toyo Kagaku Sangyo Co., Ltd. equipped with Guyaflow membrane XM300-76 manufactured by Amicon Corporation.
．． 2 Ultrafiltration was performed under a continuous airflow of Ky71om2. After the internal solution has been sufficiently concentrated, add the extra water to it, repeat the ultrapolar filtration, and repeat this operation 2 to 3 more times to obtain a concentration of 11! The filtrate and non-permeate were thoroughly removed for 1 lif.

The final retentate was completely lyophilized to obtain fraction fII6557m. Transparent? (Combine these and perform ultrafiltration in the same way using an ultrafilter equipped with Diamond 70-Menstrone YMIO-76, freeze-dry the non-permeate, and fractionate (DJ 2
93 mg was obtained. The permeate is Diaflow Membrane Y
M2 was subjected to ultrafiltration using an ultrafilter fitted with L7, and the non-permeate was lyophilized to obtain a fraction (843 mg of IID).

10 g each of the obtained fractions fIl, III and ΦD
The results of IgE inhibitory activity (1h1) on rnyb were as follows using the measurement method described in n11.

Fraction 1 has the highest IgEE production inhibitory activity among the above fractions
1. To further reduce IJ k to 141711 M, gel filtration was performed as follows.

Biogel A manufactured by Bio-Rad (1.5m (100-2
00 mesh) was equilibrated with 0.1 M Tris-HCl buffer at pM 7.6 and loaded onto a column (2,6X70α) for minutes + [! 1IfH1100m9 in the same buffer lQ
ml and added. It was bathed with the same @: buffer solution, and the bath solution was collected in 201 nl portions. The 9th to 13th fractions were collected, subjected to reduced pressure WJ Schiff, dialyzed, and lyophilized to obtain a white amorphous immunosuppressant [fraction [IJFr, 1] 25
There was one m9 child. 14-18. The ## fractions were collected and processed to give a slightly yellow-brown amorphous substance (fraction (ITlFr, 2) 10.8 rn&). Fr.
The results of measuring the IgE production inhibitory activity of , 1 and Fr, 2 were as follows.

Example 2 500 g of commercially available shelled oatmeal was coarsely ground using a coffee mill, water 21 was added thereto, and the mixture was extracted by heating in a water bath at a bath temperature of 80° C. for 4 hours. After extraction, the heel fraction fl17343, fraction (III528~) and fraction [D6777119 were obtained in the same manner as described in Example 1. The results of measuring the gE production inhibitory activity of each fraction were as follows. Ta.

DEkl Kai] Toyo Pearl 650M at pH 7.6 0.1
Equilibrate with M Tris-HCl buffer and load the column (0,26X
100 crn) and the above ([D fraction 400 In
9 was dissolved in 29 ml of the same buffer and added. 320 ml of the same buffer. Added 0.2M salt to this) 40
0In30.5M plus 400ral, 1.0
600 mg of what was added to M @ next bath, 1 volume I
Aliquots of 2Qml each were placed in test tubes. Then each test'd
UV absorption at 275 nm of phenol (liFC)
8i by tl method! It is d111 constant [2, divided into the following four fractions according to its value, rrk busy decompression,
Dialysis and freeze-drying.

1i'r, 1 No, 7-21 9.0519Fr, 2
No, 22-41 8 0.8 RQFr, 3 No.
, 42-63 5 6.8 RQFr, 4 No. 64
~100 7.2 In & IgE using each sample above
The results of measuring the suppressive ability are as follows.

The results of physicochemical analysis of the fraction 1 r, 2 thus obtained were as follows.

Elemental analysis: c 40.95chi, H5, 81%, N2.
33 g, ash content 0 g, sugar content f: 91% as total sugar content (phenol sulfuric acid method), uronic acid: galacturonic acid °C 1691r (carbazoyl jeff)
Acid method), M h't sugar nigercose, galactose, mannose or arabinose, galacturonic acid, specific rotation If: (α)” + 126.5° (C=0
．． 1%, ■) 0.01 N-NaOH solution),

BRIEF DESCRIPTION OF THE DRAWINGS: 1. The first figure shows the ultraviolet absorption spectrum of the immunosuppressant CLJ-100, and FIG. 2 shows the infrared absorption spectrum of the immunosuppressant CLJ-100.

Claims (1)

[Claims] 1) Immunosuppressant CLJ-10 having the following physical and chemical properties:
0 Elemental analysis; C39.5-41.0%, H5.8-6.
0% N2.5 or less, ash 0% Molecular suture; Approximately 100,000 yen (according to gel FJ method) Melting point: Does not show a clear melting point, and carbonizes at temperatures above 20°C. Specific optical rotation: ((Z)”+126.5° (C=0.1%
, 0.01N-NaOH aqueous solution) Ultraviolet absorption spectrum; Figure 1, outside the door If! Absorption spectrum: As shown in Figure 2, bath solubility in solvents: soluble in water, insoluble in organic solvents, color reaction: positive for iodine reaction, color and shape of substance: white amorphous substance, sugar content as nigercose, Hydrolyzed with trifluoroacetic acid, it contains mainly glucose, galactose, mannose or arabinose, galacturonic acid. is detected, but uronic acids and amine sugars are not detected. 2) Seeds of plants belonging to the genus Poaceae (Poaceae) are extracted with hot water, and components with a molecular weight of approximately 11,300,000 are fractionated from the extract using an ultrafiltration method5, and the immunosuppressant CLJ is extracted from these components.
Immunosuppressant C characterized by separating and purifying -100
LJ-100 #person method. 3) The production method according to claim 2, which is a plant belonging to the genus Colay in the family Poaceae. 4) The production method according to claim 2, wherein the hot water extraction is carried out at a temperature range of 60 to 110°C. 5) Separation and purification is carried out by one or more of the gel vEi filtration method, the ion chromatography method, and the affinity chromatography method in Claim 2 ddIIlf
! manufacturing method.