JP3254567B2 - Method for producing physiologically active substance from plant tissue and composition containing this substance - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a substance having an activity of inhibiting the production of IgE antibody and an activity of inducing interferon (INF) extracted from higher plant tissues.

[0002]

2. Description of the Related Art It is known that certain kinds of plants have the ability to produce substances having an activity of suppressing IgE antibody production. These examples are described in Perilla furutessens L . ;
Japanese J. Allergology, 41 (8), 1036, 1992), Adlay ( Coix lachryma-jobi L .; Japanese Patent Publication No. 60-130)
525) and nanban hair ( Zea mays Linne ;
19525). Further Fructus Aurantii Immaturus are not found use as herbal (Citrusaurantium L.), Dainatsume (Zizyphusjujub
a Mill. ), Shellfish ( Fritillaria thunbergii _Mig ), Thick
( Magnolia officinalis Rehd. ) And Nanten ( Nandina do
In addition to known mestica Thunb ), Kojima is one of the herbal medicines of Jumyokudokuto of Kampo formula, excluding Kawakyu, Sakura bark, Saiko, Bukuryo, Dokatsu , Licorice, Ginger, Ivy, Skull bark, Kikyo is I
It has been reported that it has gE antibody production inhibitory activity. The most active of these are Nanbange and cherry bark.

[0003] Plants belonging to the genus Susukino are widely distributed as weeds in East Asia and Southeast Asia, and 25 species are known.
Examples of typical Japanese pampas grass plants of the genus distributed in Japan, Miscanthus sinensis (M
iscanthus sinensis ), Hachijousuki ( MS var.
condensatus ), Tokiwasuki ( M. floridulus ), Ogi
( M. sacchariflorus ) and the like. Susuki is used for building materials and cow and horse materials.

[0004] Symptoms of allergy are roughly classified into four types, type I to type IV. For example, atopic bronchial asthma, allergic rhinitis, hay fever, atopic dermatitis, etc. belong to type I. It is widely known that type I allergy is deeply related to IgE antibodies, and the course until the onset of allergy is divided into the following stages. In the first stage, when an antigen enters, it stimulates the Ig to produce IgE antibodies from B lymphocytes. In the second step, the IgE antibody adheres to the surface of the mast cell, which is the target cell, and forms a sensitized state. In the third stage, when the same antigen re-enters, the IgE antibody fixed to the mast cells and the antigen are bonded in a cross-linked manner. In the fourth stage, mast cells are activated by crosslinking of antigens and antibodies,
Various biochemical reactions occur in a chain, and histamine, leukotriene, platelet-activati
Release of chemical mediators such as ngfactor (PAF). Fifth
At this stage, an inflammatory response of the tissue occurs due to the direct action of the released chemicals.

[0005] When suppressing an allergy, it is a problem which stage is to be suppressed. For example, Disodium chromoglycate (DSCG) used for the treatment of asthma, and Chinese herbal medicine Lamiaceae plant yellow garlic ( Scutellaria baicalensis Ge)
orgi ), and all currently available allergy suppressants, such as baicalen, are effective in suppressing the fourth stage. In order to fundamentally prevent or suppress allergic diseases such as asthma and hay fever, it is necessary to prevent or suppress the first-stage production of IgE antibodies, and these are being pursued in various countries around the world. However, conventionally proposed drugs also suppress the production of IgG and IgM antibodies at the same time as the IgE antibodies, and thus have a drawback of lowering the protective ability against other infectious diseases, and have not been put to practical use.

[0006]

The present invention is based on the finding that a substance isolated from the tissue of a plant of the genus Susukino has a significant IgE antibody production inhibitory activity. Therefore, an object of the present invention is to provide a method for producing a physiologically active substance from plant tissue and a composition containing this substance.

[0007]

Means for Solving the Problems According to the present invention, a gramineous family
(Gramineae ) Miscanthus ( Miscanthu )
s ), wherein the active substance is extracted from the spikes of a plant having the ability to produce a substance having an IgE antibody production inhibitory activity, and the extract is recovered from the extract. A manufacturing method is provided. Next, the present invention provides a composition comprising a physiologically active substance obtained by the method of the present invention as an active ingredient and coexisting with a pharmaceutically acceptable carrier or excipient.

Hereinafter, the present invention will be described in detail.
The raw material for the production method is a plant tissue belonging to the genus Cupressus or a variant thereof and having an activity of inhibiting IgE antibody production.

The most practical extraction method is to extract with water. A common example is extraction with 5-20 times the amount of water of the raw material, and the extraction time depends on the temperature (from room temperature to 120 ° C.).
The extraction may be of a continuous type or a batch type. However, when the pH of the extraction water is adjusted to, eg, 9-12 with an alkali such as sodium hydroxide, potassium hydroxide, or ammonium hydroxide, the extraction efficiency can be increased, and the extraction is performed at room temperature for several hours. Just leave it alone. The addition of a hydrophilic organic solvent such as ethanol can reduce the proportion of impurities, but the use of an excessively large organic solvent, for example, 50% or more, reduces the yield.

[0010] By a conventional method such as filtration, pressing, centrifugation, etc.
First, from the extract, impurities such as plant fibers are removed, and then, inactive components such as low molecular substances and pigments are removed, and the active component is recovered. A practical example is as follows. (A) For example, dialysis or ultrafiltration using a suitable membrane capable of separating substances having a molecular weight of 50,000 or more (for example, pressure of 0.1
The fractions obtained at 5-4 kg / cm ² ) are collected and lyophilized to give a brown powder. (B) The extract is optionally concentrated under reduced pressure, and a hydrophilic organic solvent (eg, methanol, ethanol, propanol, butanol, acetone, etc.) is added to the extract or its concentrate at an appropriate concentration (for example, 40 to 40% by volume). 70%), a precipitate containing the active ingredient is obtained. This can be dialyzed, for example, in a cellulose tube,
Alternatively, ultrafiltration using a membrane having a molecular weight of 50,000 or more and drying after removing the organic solvent yields a light brown powder. (C) Instead of the above organic solvent, an ammonium salt such as ammonium chloride, ammonium sulfate or cetyltrimethylammonium bromide or an inorganic metal salt such as zinc chloride or copper chloride is used in an appropriate amount (for example, 20-50 W).
/ V%) and desalting and drying in the same manner as in (b) to obtain a light brown powder. As described above, most of the raw material (90% in some cases)
The above-mentioned) can be used to recover a crude product containing the active ingredient. However, the proportion of impurities in the obtained coarse powder is (a)
Is the smallest. (A) is simple in operation, inexpensive, can be processed in a short time, and has low toxicity.

Since the active substance is a water-soluble and acidic polymer substance, it is necessary to purify the above crude product by various methods commonly used for purifying this kind of substance, for example, gel filtration or column chromatography. Can be. In the case of gel filtration, elution may be carried out with an appropriate buffer, but usually with water. Purification can also be carried out using a suitable anion or cation ion exchange cellulose and a buffer.

Examples of practical gel filtration agents are Sephadex G-50 to G-200, Sepharose 2B to 6B, Sephacryl S-200 or S-300.
(Pharmacia Fine Chemical AB, Sweden
Examples of practical ion-exchange celluloses include Biogel A (manufactured by Bio-Rad Laboratories, USA), Biogels P-30 to P-300, and DEAE Sephadex A-25 and A-50 (Cl ^- type), Q
AE Sephadex C-25 and C-50 (Cl ^- type)
, CM Sephadex C-25 and C-50 (Na ⁺
Mold), SP Sephadex C-25 and C-50
(Na ⁺ type), DEAE Sephacell (Cl ⁻ type), DE
AE Sepharose CL-6B (Na ⁺ type) (manufactured by Pharmacia Fine Chemical AB, Sweden). If desired, impurities can be further removed by combining the above-mentioned purification methods.

Physicochemical properties The physicochemical properties of the active substance purified by the method described in Example 1 are as follows. 1) Appearance Light brown amorphous powder 2) Elemental analysis H: 4.20%, C: 40.61
%, N: 2.51%. 3) Molecular weight from about 50,000 to about 1,000,000. 4) Melting point or decomposition point Unclear melting point, carbonized at about 210 ° C. Stable at 100 ° C. 5) Ultraviolet absorption spectrum As shown in FIG.
In NaOH). 6) Infrared absorption spectrum As shown in FIG. 2 (KBr
Law). 7) Solubility in various solvents Dissolves in water. It dissolves well in particularly alkaline aqueous solutions such as potassium hydroxide, sodium hydroxide, ammonium hydroxide and potassium carbonate. Poorly soluble in methanol, ethanol, butanol, acetone, chloroform and ether. 8) Color reaction Positive for ninhydrin reaction and phenol / sulfuric acid reaction. 9) Properties acidic, pH 6.4 10) Specific rotation

[0014]

(Equation 1)

Biological Characteristics Using the sample of the active substance of the genus Cupressus purified by the method described in Example 1 described below, Test Example 1 (a) described below was used.
The biological properties measured by the method (b) are as shown in Table 1, and the activity of inhibiting the production of IgE antibody was confirmed. The results of the IgE antibody production suppression test in the secondary antigen immune response are shown in Table 2, and the suppression activity was observed.

[0016] Of the same Gramineae plants, the spikes of the plant reed ( Phragmitescommunis Trin ), which is close to the genus Susukino, and the genus Juzudama ( Coix lachryma-jobi L. )
Female spikelets and Imperata cylindrica
var. major ), a component having an IgE antibody production inhibitory activity was not detected. Tricine (according to the class of the herb) and purinin and miscantoside (according to folk medicines of plants and animals) are extracted from the lanceolus of the genus Susukino, and they show neither IgE antibody production inhibitory activity nor interferon inducing activity.

[0017]

[Table 1]

[0018]

[Table 2]

Interferon-inducing activity Using a sample of an active substance purified by the method described in Example 1 described below, the result of measurement by the method of Test Example 2 (a) described below is shown in Table 3, and the results are shown in Table 3. Was observed. The interferon produced was alpha-type, similar to that induced with endotoxin (endotoxin), and peaked at 2 hours in mouse blood.

[0020]

TABLE 3 Serum IFN production in mice 血清 Injection volume Blood sampling Time and serum IFN value (IU / ml) (mg / kg) 0 1 2 3 5 hr ───────────────────────────── ８ 8 mg <10 51.0 ± 3.2 153.0 ± 19.4 29.2 ± 2.4 21.4 ± 2.2 ─────────────────────────── ─────────

In order to effectively administer the physiologically active substance of the present invention to humans and animals, the composition provided by the present invention comprises a physiologically active substance obtained by the method of the present invention as an active ingredient, Characterized by being co-existed with a carrier or excipient that is acceptable. The composition of the present invention can be in a form suitable for oral, injection, transdermal, intramucosal administration and the like. For example, compositions for oral administration can be solid or liquid, powders, syrups, capsules, granules,
Emulsions, suspensions, drops and the like may be used. Carriers or excipients for such compositions are well known. Examples of excipients for tablets are lactose, potato and soluble starch, magnesium stearate and the like, and examples of carriers for injection are sterile water, physiological saline, almond oil and the like. Or may be added to the active substance before use.

If desired, the composition may further comprise conventional materials such as binders, stabilizers, emulsifiers, suspending agents, dispersants, lubricants, preservatives, extenders and the like. Practical composition forms include, for example, buccal, troche, eye drops, nasal drops, suppositories, injections, inhalants, sprays, ointments, plasters, liniments, films, baths and the like.

Advantageously, these compositions are prepared as a dosage unit, shaped so as to provide a fixed amount of the active substance. Suitable examples are tablets, coated tablets, ampules, capsules, suppositories. A practical example of an active substance contained in a dosage unit is a unit content for intravenous injection of 1 unit.
If, for example, an oral administration agent is about 10-100
Fold, about 2-5 times for subcutaneous injection, and about 1.times. For intramuscular injection.
5-5 times, about 2-10 times for buccal and troche, about 4-5 times for suppositories, and 1-5 times for sprays. The amount to be administered intravenously to humans or animals varies depending on the type, age and purpose of administration of the host, but may be, for example, 0.1 to 10 mg / kg. For example, even when a large amount of the crude product obtained by the method (a) was orally administered to animals as it was, no remarkable side effects were observed.

[0024]

Example 1 Dried Miscanthus sinensis An
derss ) spikelets (1kg), shredded and washed twice with water,
Water (10 l) was added, and the mixture was heated and extracted at 95 ° ± 5 ° C. for 1 hour. This was filtered to remove the residue, and a brownish transparent extract was obtained. The yield when freeze-dried was 0.72% based on the starting material. This is distilled under reduced pressure and the dialysis membrane
(mwco: 50,000; US Spectrum Medical, Inc.)
(Industries Incorporated) to obtain a fraction having a molecular weight of 50,000 or more. This was freeze-dried to obtain a light brown powder (6.07 g). The IgE antibody production inhibitory activity of the non-dialysis fraction increased about 20-fold (see Table 1). DEAE Toyo Pearl 650 (Toyo Soda Co., Ltd.)
Equilibrated with H7.4 0.1 M Tris-HCl buffer,
Packed in a 2.6 × 70 cm column. 200 mg of the above non-dialysis fraction (molecular weight: 50,000 or more) was dissolved in 10 ml of the same buffer, added to the column, and then eluted with the same buffer.
Aliquots were collected by ml. After collecting the fractions from No. 7 to No. 20, 0.2M sodium chloride was added to the eluate and eluted similarly. The fractions from No. 40 to No. 50 were collected, and the molecular weight of the fraction was limited to 10,000. After desalting using a filtration membrane (Diaflow Membrane PM-10 manufactured by Amicon, USA), the solution was freeze-dried to obtain a light brown amorphous powder (32.2 mg). Its IgE antibody production inhibitory activity was about 100 times or more that of the crude extract. The acute toxicity (LD ₅₀ ) was about 400 mg / kg (mouse).

[0025]

Example 2 Dried Hachijousuki flower spikes (100
g) was cut into small pieces, water (2000 ml) was added, 1N sodium hydroxide was added dropwise with stirring to adjust the pH to 11.0, and the mixture was allowed to stand at room temperature for 2 hours to obtain a filtrate (1900 ml). This is 1N
The pH was adjusted to 7.0 with hydrochloric acid, and the mixture was subjected to high-speed centrifugation at 10,000 g for 30 minutes to obtain a brown transparent extract. This was treated with an ultrafilter UHP-76 type (manufactured by Toyo Kagaku Sangyo) using a Diaflow Membrane PM-50 ultrafiltration membrane (fraction molecular weight: 50,000; manufactured by Amicon, USA), Similarly, treatment by column chromatography and freeze-drying gave a light brown amorphous powder (30.3 mg). Its IgE antibody production inhibitory activity was slightly lower than that of Example 1.

[0026]

Example 3 Dried flounder spikes (400 g) were cut into small pieces, added with water (8000 ml), extracted with hot water at 80 ° ± 3 ° C. for 2 hours, filtered to remove plant fibers, and removed from Diaflow membrane XM. The mixture was treated with a -50A ultrafiltration membrane (fraction molecular weight: 50,000; manufactured by Amicon, USA), and the residue was freeze-dried to obtain a light brown powder (2.45 g). According to Example 1, this powder was treated by column chromatography using DEAD-Toyopearl and then freeze-dried. Although the yield of the product was lower than that of Example 1, the activity of inhibiting the production of IgE antibody was not significantly different.

[0027]

Example 4 The spikes (100 g) of dried ogi ( M. sacchariflorus ) were cut into small pieces, and water (2000 ml) was added.
The mixture was extracted at 0 ° ± 3 ° C. for 2 hours and treated according to the method described in Example 2 to obtain a light brown amorphous powder (26.4 mg).
Its IgE antibody inhibitory activity was slightly lower than that of Susukino extract.

Test Example 1 Inhibition of IgE Antibody Production (a) Production of IgE Antibody The active substance obtained by the method described in Example 1 was dissolved in physiological saline in the amount shown in Table 1, and the solution was diluted to 6 weeks of age. BALB / c mice (female, 5 mice per group) are injected intraperitoneally by 0.5 ml.
After 24 hours, 10 μg of 2,4-dinitrophenyl ovalbumin as an allergen (in 0.5 ml) per mouse
Is administered intraperitoneally together with a suspension of 2 mg of aluminum hydroxide gel. As a control, the same mouse without sample administration (5 per group)
), The same antigen was administered together with an adjuvant, 14 days after the administration of the antigen, the mice were bled by cardiac stenosis, the blood of each of the 5 mice was mixed, the serum was separated, and the serum was separated with saline for 10 or 3 days.
Measurement samples were prepared by diluting 0, 100 and 300 times.

(B) PCA method (Passive cutaneous ana
Measurement of IgE activity by phylaxis Male rats weighing about 180 g (Wister species; 5 mice per group) were shaved on the back and 0.1 ml of the diluted serum of the above immunized mouse was injected into the skin. 20 animals can be administered per animal. 4 hours after injection, 10 mg of dinitrophenyl egg albumin antigen
Is dissolved in 10 ml of 1% Evans blue solution, and 1 ml per rat is injected into the tail vein. Thirty minutes after the injection, the rats are anesthetized with chloroform, and the skin on the back is peeled off, and the presence or absence of blue spot reaction and the size are measured from the back.
When the inhibitory effect was sufficient and no IgE antibody was present in the serum, no blue spots were observed in each dilution of the serum, and when there was an IgE antibody presence reaction, the skin site of the serum injection had a diameter of 5 mm.
A remarkable blue spot of about 20 mm or more appears. 4 when no blue spots appeared, 3 when the blue spots appeared as a partial arc around the injection site, 2 for blue spots with a diameter of 10 mm or less, 1 and 20 mm for blue spots of 11 to 19 mm The score was shown with the above blue spot as 0.

Test Example 2 IFN Induction Method and IFN Activity Measurement Method (Reference: NB
Finter, Interferonsand Interferon Inducers, North-
(Holland Pub. Co., p.46, p.142, 1973) (a) Method for Inducing IFN by In Vivo Method Aqueous solution (1) of the present active substance obtained by the method described in Example 1
mg / ml) in BALB mice (6 weeks old; 5 per group)
, And blood was collected from the heart 1, 2, 3 and 5 hours later, each blood was pooled, and the serum was subjected to IFN activity measurement.

(B) The activity of the produced IFN was measured by the 50% CPE half reduction method. The cells used were LY cells derived from mouse fibroblasts, and the culture was diluted with Eagle's MEM containing 5% FCS to dilute the sample, and the cells were treated at 37 ° C. for 1 day, and vesicular stomatis was used.・ Virus (VSV) is used to attack virus (MOI =
0.1). The unit of IFN activity is CPE5
Shown as the reciprocal of the maximum dilution of the sample required to reduce by 0%. (C) The serum obtained from the mouse injected with the present active sample exhibited interferon properties such as species-specificity, virus non-specificity, instability upon heating at 56 ° C. for 30 minutes, and inactivation of trypsin treatment. .

Formulation Examples (1) Injection 1.0 ml of physiological saline 10 mg of active substance Aseptically encapsulated in 2 ml ampules.

(2) troche hydroxypropylcellulose 5.2 g vinylpyrrolidone 4.5 g active substance 0.3 g

(3) Suppositories polyethylene glycol 400 0.8 g liquid polyethylene glycol 1500 0.2 g active substance 0.2 g

(4) Syrup CMC-Na 0.2 g Single syrup 20 g Na cyclamate 0.1 g Ethyl paraffin 0.04 g Active substance 0.1 g

(5) Ointment Purified lanolin 5 g Beeswax 5 g White vaseline 87 g Active substance 3 g

(6) Coating agent Calcium hydroxide 0.3 g Glycerin 20 ml Ethanol 25 ml Active substance 2.5 g Make up to 100 ml with water.

(7) Nasal drops Methyl parabenzoate 0.3 g Propyl parabenzoate 0.2 g Tetrahydrozoline hydrochloride 1.0 g Physiological saline Suitable active substance 3.0 g

(8) Film agent Pullulan 95.0 g Active substance 0.2 g

[0040]

The raw material for producing the present active substance is widely distributed as a weed in the wilderness, so that it can be produced in a simple manner and at low cost, and has the highest activity of conventional plant origin. It was found to be approximately equivalent to the activity of the substance.
The active substance is used as a primary and secondary immune response to Ig
In addition to suppressing E antibody production, it has IFN-inducing activity,
Since it does not affect the production of IgG and IgM antibodies, which are important for the prevention of infectious diseases, human type I allergies (bronchial asthma,
It is expected to be useful not only for prevention of hay fever, atopic dermatitis, etc.) and improvement of symptoms, but also for enhancement of non-specific defense as IFN activity.

[Brief description of the drawings]

FIG. 1 is an ultraviolet absorption spectrum (in 0.1 N NaOH) of an active substance obtained by the method described in Example 1.

FIG. 2 is an infrared absorption spectrum (KBr method) of an active substance obtained by the method described in Example 1.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) A61K 35/78 BIOSIS (DIALOG) CA (STN) MEDLINE (STN)

Claims (5)

(57) [Claims] 1. A flower of a plant belonging to the genus Gramineae and belonging to the genus Miscanthus and capable of producing a substance having an IgE antibody production inhibitory activity.
A method for producing a physiologically active substance, comprising extracting the active substance from ears and collecting the active substance from the extract. 2. The method according to claim 1, wherein the physiologically active substance further has an interferon-inducing activity. 3. The method according to claim 1, wherein the extraction is carried out with water. 4. The method according to claim 3 , wherein the extraction is performed at pH 9-12. 5. A flower of a plant belonging to the genus Gramineae and belonging to the genus Miscanthus and having the ability to produce a substance having an IgE antibody production inhibitory activity.
A composition comprising, as an active ingredient, a biologically active substance obtained by a method comprising a step of extracting the above-mentioned active substance from ears and recovering the same from the extract, and coexisting with a pharmaceutically acceptable carrier or excipient. object.