JPH01224317A - Cellular antibody production inhibitor - Google Patents

Abstract

PURPOSE:To provide a cellular antibody production inhibitor having a cellular antibody production-inhibiting action and effective against chronic hepatitis, nephrotic syndrome, etc., by comprising phellodendrine or a salt thereof as an active ingredient. CONSTITUTION:A cellular antibody production inhibitor comprises phellodendrine of the formula or a salt thereof as an active ingredient. The compound of the formula is obtained by extracting a galenical Phellodendron amurense with methanol, concentrating the extract, dissolving the concentrated extract in water, shaking the solution together with ethyl acetate, distributing the shaken solution, subjecting the distributed ethyl acetate solution to a column chromatography to provide a crude fraction and further repeatedly subjecting the solution to the column chromatography. The galenical Phellodendron amurense is the periderm-free bark of Phellodendron amurense RUPR. of Rutaceae or the same genus plant and has been used from old times for spasmolytic, stomachine and antibacterial purposes.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to therapeutic agents for diseases involving cellular antibodies.

[Prior Art and Problems] Diseases involving cellular antibodies, such as chronic hepatitis, infectious or mixed asthma, and nephrotic syndrome, have a large number of patients and are of great social interest.

Conventionally, steroid drugs and the like that suppress cell-mediated antibody production have been used to treat these diseases, but their use is restricted due to their many side effects. Therefore, it has been desired to develop a cell-mediated antibody production inhibitor that has few side effects and excellent medicinal efficacy.

[Means for Solving the Problems] In order to provide an inhibitor of cellular antibody production that is effective in treating diseases such as chronic hepatitis and nephrotic syndrome, the present inventors have determined the inhibitory effect of cellular antibody production on extracts of the herbal medicine Huangbai. As a result of screening, it was discovered that ferrodendrin, a component of yellow oak, has a strong inhibitory effect on cell-mediated antibody production.

The present invention is based on this knowledge, and is a cellular antibody production inhibitor containing ferrodendrin or a pharmacologically acceptable salt of ferrodendrin as an active ingredient.

Yellow oak is a member of the Rutaceae family (Rutaceae).
hellodendron amurense RUP
R,) or other congenerous plants, and is a herbal medicine that has been used since ancient times for antispasmodic, stomachic, and antibacterial purposes.

Ferrodendrin is a compound extracted and isolated from this yellow oak, and is represented by the following structural formula.

Ferrodendrin can be obtained from Huangbai, for example, in the following manner.

The methanol extract obtained by extracting and concentrating Aspergillus cypress with methanol is dissolved in water and shaken and distributed with ethyl acetate. After partitioning, the aqueous layer was concentrated and M(l gel CHP 20 P,
Column chromatography is performed using a porous gel such as Sephadex LI-(20) as a carrier, and sequential elution is performed with water, water-methanol, methanol, and hydrochloric acid-methanol. From the water-methanol eluate, ferrodendrin,
A crude fraction containing magnoflorin and phenols is obtained.

This crude fraction was subjected to column chromatography again,
Elute with a mixed solvent of acetic acid-water-methanol, and obtain both fractions containing ferrodendrin from the eluate.

Furthermore, both fractions were subjected to column chromatography and eluted sequentially with water, water-methanol, and methanol. Crude ferrodendrin is obtained from the water-methanol eluate.

Next, the crude ferrodendrin is subjected to column chromatography, and the eluate obtained by elution with ethanol is distilled off, and then recrystallized from acetone-methanol to obtain ferrodendrin.

A specific example of the production of ferrodendrin is shown below.

Specific Example 1 2 kg of yellow oak was extracted four times with 8Q methanol, and the obtained extracts were mixed and concentrated under reduced pressure to obtain 360 g of methanol extract. Dissolve the extracted extract in water lσ,
After extraction three times with ethyl acetate 5007, the aqueous layer was concentrated under reduced pressure to obtain 280 g of a water-soluble portion. MCI gel C
The mixture was subjected to column chromatography using HP 20 P and the mixing ratio of water and water:methanol was increased sequentially.Water = methanol, methanol, hydrochloric acid-methanol (2:9
8), fractionated in 120-unit portions, mixed the eluted portions of water-methanol (7:3) and water-methanol (3:2), and concentrated under reduced pressure to obtain brown powder ferrodendrin and 60 g of a crude fraction containing phenols was obtained.

This progenitor 602 was subjected to column chromatography using MCI gel CHP20P, and acetic acid-water (2:98
), acetic acid-water-methanol, acetic acid-methanol (2:98) in which the mixing ratio of acetic acid: water: methanol was increased sequentially
The mixture was developed in the following order, separated into 120-units, and the eluates obtained by elution from acetic acid-water-methanol (2:88:1 o) to acetic acid-water-methanol (2:58:40) were mixed. ,
The mixture was concentrated under reduced pressure to obtain 12 g of both components containing brown powder ferrodendrin.

Furthermore, the fraction 129 was subjected to Fuji gel 0DS
It was subjected to column chromatography using G3, and developed in the order of water, methanol, and methanol in which the mixing ratio of water and water:methanol was increased sequentially, and 20-methanol fractions were collected.
The eluted portions of methanol (4:l) and water-methanol (3:2) were mixed and concentrated under reduced pressure to obtain crude ferrodendrin 3.
I got a 9.

Next, add 3g of crude ferrodendrin to Sephadex LH20.
The eluted portions of ethanol IQ were combined, concentrated under reduced pressure, and crystallized from acetone-methanol to obtain 2.19 colorless needle crystals. The physicochemical properties of this colorless needle crystal were as follows, and from these properties it was determined that it was ferrodendrin acetate.

Melting point = 205-208℃ Specific rotation: [α]'5 118.9゜(c-0.4,
Hto) Infrared absorption spectrum νKBtα-1:3400.16
20.1400 Proton nuclear magnetic resonance spectrum (δPpm 111 CD30D): 1.88 (3H, s), 3.21 (3H, s).

2.8-3.6 (4H, m).

3.85, 3.86 (each3H,s).

3.85 (2H, m).

4. 5 5. 4. 7 3 (Eachl H, d, J = 15 Hz).

4.7 5 (I H, m), ”6.6 5.6
．． 7 1,6.7 5,6.8 6(each l
H, s) Specific Example 2 Ferrodendrin acetate obtained in Specific Example 1 was dissolved in a small amount of water, and acidified with a 1% aqueous hydrochloric acid solution. This was concentrated under reduced pressure to obtain a light brown amorphous powder. The physicochemical properties of this light brown amorphous powder are as follows, and from these properties it was determined that it was chlorinated ferrodendrin.

Specific optical rotation = [α coIs -136,4° (c = 0.
41, C) [30H) Infrared absorption spectrum ν22
cM-': 3400.1610.1520 Specific Example 3 Ferrodendrin chloride obtained in Specific Example 2 was dissolved in a small amount of water, and a concentrated aqueous potassium iodide solution was added to obtain a brown precipitate.
This was collected by filtration and methanol-ethanol-acetone (1
:1:2) to obtain colorless needle crystals. The physicochemical properties of this colorless needle crystal were as follows, and based on these properties, it was determined that it was brominated ferrodendrin.

Melting point: 257-259°C Specific rotation ratio variation [α]': -125,8° (c=0
．． 41, CH30H) Infrared absorption spectrum νvaa: ('717-'
:3200.1620.1530 Next, the fact that ferrodendrin has an inhibitory effect on cellular antibody production will be explained using experimental examples.

Experimental Example 1 The abdomen of a ddY male mouse (8 weeks old) was shaved, and 1% cyclyl chloride ethanol solution 0.0% and I RQ was applied.
Sensitized. Six days later, 1% chlorinated cyclyl olive oil solution + 5 d each was applied to the front and back surfaces of both earlobes for induction (-second induction). After 24 hours, the earlobe swelling was measured using a dial thickness gauge.
). The measured value obtained by subtracting the measured value of the induced surface is considered to be the swollen dust due to the primary immune reaction,
It was used as an indicator of the inhibitory effect on cellular antibody production.

Two days later, the mice were sensitized in the same manner as above, and 6 days later they were induced (secondary induction), and earlobe swelling was measured using a Dial Sickness Geno 24 hours later as before. The value obtained before induction was subtracted from the measured value to determine the swelling caused by the secondary immune reaction, and was used as an index of the inhibitory effect on cell-mediated antibody production.

In addition, a physiological saline solution of ferrodendrin was intraperitoneally administered from the first sensitization mortar for 5 days, and only physiological saline was administered to controls. The swollen dust in the primary immune reaction and the secondary immune reaction are as shown in Table 1, respectively.

Table 1 Also, in Experimental Example 1, when ferrodendrin was administered twice, once immediately before the secondary induction and once 16 hours later, the second
As shown in the table, no change was observed in the swollen dust in the earlobes, confirming that ferrodendrin suppresses cellular antibody production.

Table 2 Experimental Example 2 Balb/c mice and CBF The spleens of the mice were removed aseptically and treated with Hang's balanced salt solution
A cell suspension was obtained through a stainless steel wire mesh (200 mesh) in a balanced salt solution (5 solution).

Next, an appropriate amount of 0.75% ammonium chloride solution was added to lyse the red blood cells in the cell suspension, washed three times with Hang's balanced salt solution, and adjusted to 10' cells/Id.

CBF, Ba1b/c mouse tile cell suspension 0.05- to the right hind foot footprint of the mouse, CBF, mouse tile cell suspension 0.05- to the left hind foot paw print. Each OJd was injected subcutaneously. Eight days later, the axillary lymph nodes of both legs were removed and their weights were measured. The weight of the left axillary lymph node was subtracted from the weight of the right axillary lymph node to determine the strength of the graft-versus-host reaction.

Ferrodendrin was dissolved in physiological saline for intraperitoneal administration and in purified water for oral administration, and 8 days from the day of cell transfer.
It was administered for days. In addition, only the solvent was administered to each control. Table 3 shows the intensity of the graft-versus-host reaction when ferrodendrin was administered intraperitoneally and orally.

Table 3 Experimental Example 3 A full thickness of skin with a diameter of 1 L:11 was removed from the dorsal midline of a C57BL/6 mouse under bendoparbital anesthesia to prepare a transplant bed. The skin of a C3II/He mouse of the same size, which was aseptically collected as a supply skin piece, was transplanted to this, and detachment of the skin piece was observed. Ferrodendrin was administered intraperitoneally for 11 days starting 3 days before transplantation, and only physiological saline was administered to controls.

As a result, the survival time of the control skin graft was 8.7
9 and 101 days and 5 mg/day of ferrodendrin
! The engraftment time of the grafted skin in the 97 and 9 administration groups was 9.9, respectively.
5 days and 9.6 days.

These results confirmed that ferrodendrin has an excellent inhibitory effect on cell-mediated antibody production and allograft rejection. This ferrodendrin is effective for mixed asthma, chronic hepatitis, nephrotic syndrome, sarcoidosis,
It can be used to treat diseases involving cellular antibodies such as Behcet's disease.

Further, in these experiments, no side effects were observed due to the administration of ferrodendrin.

Furthermore, ferrodendrin was dissolved in physiological saline for intraperitoneal administration, and purified water for oral administration, and each was administered to mice (12 mice), and the L was determined to be alive or dead after 72 hours. Ds. Value (Litchrield-w
The safety of ferrodendrin was confirmed by calculation of 140 R9/9 for intraperitoneal administration and 9 for 197 for oral administration.

Next, the dosage and formulation of ferrodendrin will be explained.

Since ferrodendrin itself has medicinal effects, any salt may be used as long as it is pharmacologically acceptable. Specific examples of salts include acetates, chlorides, bromides, and the like.

Ferrodendrin can be administered to animals and humans either neat or with conventional pharmaceutical carriers. The dosage form is not particularly limited and may be selected and used as appropriate, and may include oral dosage forms such as tablets, capsules, granules, fine granules, and powders, and parenteral dosage forms such as injections and halved tablets. It will be done.

In order to achieve the desired effect as an oral agent, it is necessary to administer 5 to 500 txy of ferrodendrin in divided doses several times a day for adults, depending on the patient's age, weight, and degree of disease. It seems appropriate to take it.

Oral preparations such as tablets, capsules, and granules of ferrodendrin are manufactured according to conventional methods using, for example, starch, lactose, sucrose, mannitol, carboxymethyl cellulose, cornstarch, inorganic salts, and the like.

In this type of preparation, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, perfumes, etc. can be used as appropriate in the excipients. Specific examples of each are shown below.

[Binder co-starch, dextrin, gum arabic powder, gelatin,
Hydroxypropyl starch, methylcellulose, sodium carboxymethylcellulose, hydroquinopropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, macrogol.

[Disintegrant] Starch, hydroquinepropyl starch, sodium carboxymethylcellulose, sodium carboxymethylcellulose, carboxymethylcellulose, low substituted hydroquinepropylcellulose.

[Surfactant] Sodium lauryl sulfate, soybean lentin, polysaccharide fatty acid ester, polysorbate 80° [Lubricant] Talc, waxes, hydrogenated vegetable oil, polysaccharide fatty acid ester, magnenium stearate, calcium stearate, aluminum stearate, Polyethylene glycol.

[Fluidity promoter] Light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate.

In addition, ferrodendrin can be used in suspensions, emulsions,
It can also be administered as a tablet or elixir, and these various agent layers may contain flavoring agents and coloring agents.

In order to exert the desired effect as a parenteral agent, the amount of ferrodendrin for an adult is usually 0.5 to 100% per day, although it varies depending on the patient's age, weight, and severity of the disease. Intravenous injection up to 00■, intravenous drip infusion, subcutaneous injection, and intramuscular injection are considered appropriate.

This parenteral preparation is manufactured according to a conventional method, and generally uses distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol, etc. as a diluent. be able to. Furthermore, a bactericide, a preservative, and a stabilizer may be added as necessary. In addition, from the viewpoint of stability, this parenteral preparation may be filled into a vial or the like, then frozen, water removed by ordinary freeze-drying techniques, and a liquid preparation prepared from the freeze-dried product immediately before use. Furthermore, isotonizing agents, stabilizers, preservatives, soothing agents, etc. may be added as appropriate.

Other parenteral preparations include liquid preparations for external use, liniments such as ointments, and halved preparations for intrarectal administration, and are manufactured according to conventional methods.

Example 1 ■Corn starch 44g ■Crystalline cellulose 40g ■Carboxymethyl cellulose calcium 59 ■Light anhydrous silicic acid 0.59■Magnesium stearate 0.59■Ferrodendrin
To9 total 100g According to the above recipe, ■ to ■ were mixed uniformly and compression molded using a key press to obtain one tablet of 200 M9.

These tablets contain Compound 2 (J19), and adults should take 5 to 25 tablets a day in several doses.

Example 2 ■ Crystalline cellulose 84.59 ■ Magnesium stearate 0.59 ■ Carboquine methylcellulose calcium 59 ■ Ferrodendrin IOg meter 1
009 According to the above recipe, mix ■, ■, and part of ■ uniformly, compression mold, crush, add remaining amounts of ■ and ■, mix, and compression mold with a key press to make a portion of 200 m9 tablets were obtained.

This tablet-tablet contains the compound 20m9,
For adults, take 5 to 25 tablets a day in several divided doses.

Example 3 ■ Crystalline cellulose 34.59 ■ 10% hydroxypropyl cellulose ethanol solution 509 ■ Calcium carboxymethyl cellulose 52 ■ Magnesium stearate 0°5 g ■ Ferrodendrin 109 total 1009 According to the above recipe, ■, ■, and ■ were uniformly poured. After combining the ingredients, granulating them using an extrusion granulator, drying and crushing them, and compressing them using a tablet machine, they are compressed into tablets of 200 or more. Obtained.

This tablet-tablet contains the compound 20m9,
For adults, take 5 to 25 tablets a day in several divided doses.

Example 4 ■Corn starch 84g■Magnesium stearate 0.59■Calcium carboxymethyl cellulose 59■Light anhydrous silicic acid 0.59■Ferrodendrin 10g Total 100y Mix ■~■ uniformly according to the above recipe and compress with a compression molding machine After molding, the mixture was crushed using a crusher and sieved to obtain granules.

This granule 19 contains compound 100119, and is taken by adults in doses of 0.5 to 5 g in several doses per day.

Example 5 ■ Crystalline cellulose 409 ■ 1O% hydroxypropyl cellulose ethanol solution 509 ■ Ferrodendrin 1M Total 100 g According to the above recipe, ■ to ■ were uniformly mixed and then wetted. After going into an extrusion granulator and granulating, the mixture was dried and sieved to obtain granules.

This granule N) contains 100 to 100 of the compound, and is taken by adults in 0.5 to 59 doses per day in several doses.

Example 6 ■Corn starch 895g ■Light silicic anhydride 0.59■Ferrodendrin 10g Total 10 (M) Mix ■~■ uniformly according to the above recipe, 200 t
q was filled into a No. 2 capsule.

One capsule of this preparation contains Compound 2 (1@), and adults should take 5 to 25 capsules a day in several doses.

Example 7 ■ Distilled water for injection Appropriate amount ■ Glucose 200 Q ■ Ferrodendrin 50! ir9 total amount
5d After dissolving ■ and ■ in distilled water for injection, they were injected into a 5d ampoule and autoclaved at 121'C for 15 minutes to obtain an injection.

Claims (1)

[Claims] A cellular antibody production inhibitor containing ferrodendrin or a pharmacologically acceptable salt of ferrodendrin as an active ingredient.