JPH0930983A - Apoptosis inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an apoptosis inhibitor having inhibitory action on apoptosis, useful for treating a hepatosis such as viral hepatitis, liver cirrhosis, etc., and a diseases, in which apoptosis participates, such as reduction in immunological function during stress and alleviating symptoms. SOLUTION: This apoptosis inhibitor contains at least one of berberine, palmatine, capillarisisn, 7-methylcapillarisisn, 6,7-dimethylescuretin, arcapillin, capillin, capillene and capirallin. components contained in OUREN (rhizome of Coptis japonica) and INNTINNKOU (whole grass of Artemisia capillaris).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an apoptosis inhibitor which can be used as a medicine such as an immunomodulator and a hepatitis therapeutic agent.

[0002]

2. Description of the Related Art In recent years, a mode of apoptosis (also referred to as apoptosis; self-destruction or cell self-destruction) has been found with respect to the death of cell tissues, and has attracted attention.

Unlike necrosis, which is a pathological cell death, this apoptosis is considered to be a death that is originally incorporated in the gene of the cell itself. That is, some external or internal factor is triggered to activate the gene that programs apoptosis, and the programmed death protein is biosynthesized based on this gene, and the generated programmed death protein decomposes the cell itself and causes death. Is believed to reach.

[0004] Due to the nature of apoptosis, if apoptosis can be expressed in a desired tissue or cell, unnecessary cells or pathogenic cells can be naturally eliminated from the living body. Therefore, conventionally, the search and development of compounds having an apoptosis-inducing action have been extensively conducted. For example, the present applicant has proposed several apoptosis inducers so far (Japanese Patent Laid-Open No. 5-331).
061, JP-A-6-25002, JP-A-6-26
-72888).

However, in recent years, it has been reported that suppressing apoptosis is useful. For example,
It is as shown below. Preventing neuronal cell loss in patients such as Alzheimer's, HI
To prevent T cells not infected with V from dying by apoptosis, and to prevent cell death of normal cells that are easily damaged by side effects such as anticancer agents.

However, there are few compounds known to be able to suppress apoptosis in the past, and there is a demand for the development of a compound having a more excellent apoptosis suppressing action.

[0007]

The present invention has been made in view of the above points, and has an action of suppressing apoptosis, for example, liver diseases such as viral hepatitis and cirrhosis, and immunity during stress. Provided is an apoptosis inhibitor useful for treating diseases associated with apoptosis such as hypofunction and alleviating symptoms.

[0008]

The present invention is firstly selected from the group consisting of berberine, palmatine, capillarisin, 7-methylcapillarysin, 6,7-dimethylesculetin, alcapillin, capillin, capylene and capillarine. The present invention provides an apoptosis inhibitor containing at least one of these as an active ingredient.

Secondly, the present invention relates to yellow lanterns, yellow oak, soybeans, red calyx, cherry bark, shreds, poems, maranko, roads, rush, makigashi, mahuang, sardines, frankincense, Provided is an apoptosis inhibitor containing at least one herbal medicine selected from the group consisting of Pleurotus sinensis, Root of radix, and Pinus chinensis.

Thirdly, the present invention relates to Orengedokuto, Sanohreishinto, Keishibukuryomaru, Keishikashakuyakudaioto, Daijokito, Kachinchinto, Rokumimaru, Kibosaito. , Asako Ninmaru, Gyoreito, Tokikenchuto, Kawakyucha Sansan, Keishikaryukotsu Oyakuto, Kureiyuto, Hofutsushosan, Tokokujokito, Jitsubori, Daio Botanpi Provided is an apoptosis inhibitor containing one kind of a herbal medicine selected from the group consisting of hot water, Saibokuto, Mao-to, Mao-bushi-saishin-to, and Chichin-Goreisan.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The first invention of the present application is berberine and palmati.
ne), capillarisin (capillarisi)
n), 7-methylcapillarycin (7-methylc
apillarisin), 6,7-dimethylesculetin (6,7-dimethylscureti)
n), alcapillin, capillin, capilene
ne) and capillarin (capiralin) is an apoptosis inhibitor containing at least one compound selected from the group consisting of.

Each of these active compounds is a component contained in crude drugs such as chinen chinensis, yellow liquor and yellow oak. For example, berberine and palmatine are the constituents of yellow reed. Among these, capillaryin, 7-methylcapillin, 6,7-dimethylesculetin, alcapillin,
Capillin, capylene, and capillarin are all ingredients contained in Kachinoki. Further, 6,7-dimethylesculetin is also a component contained in flower roots.

The effective compound of the apoptosis inhibitor of the first invention of the present application can be used by extracting and separating it from a crude drug containing the effective compound. In addition, an artificially synthesized product can also be used.

Further, the apoptosis inhibitor of the first invention of the present application may contain a crude drug containing the above-mentioned effective compound and an extract of the crude drug. Specifically, for example, at least one crude drug and a crude drug extract selected from the group consisting of Huanglian, Huangba, and Amaranthus chinensis can be used.

Furthermore, the apoptosis inhibitor of the first invention of the present application also includes Chinese herbs containing the above-mentioned effective compounds.
Specifically, for example, Oren-gedoku-to, San-rei-shin-to, Aoi-chinka-to, Gyorei-to, Hofutsu-sei-san, Saiboku-to, Sai-rei-to, Oren-to or Go-chin-gorei-san. . Further, the apoptosis inhibitor of the first invention of the present application also includes a composition containing the extract of the above Chinese herb medicine. Further, the apoptosis inhibitor of the first invention of the present application may be a composition containing an extract from a natural product such as a plant containing the above-mentioned effective compound. The second invention of the present application is Yellow Ren, Yellow Kashiwa, Suiki, Red Qinkyu, Sakura peel,
It is an apoptosis inhibitor containing at least one kind selected from the group consisting of 樸 荟, 虸, 蜸 子, Maranko, Rojidori, Yakuso, Makigashi, Mao, 苯 苓, frankincense, Maeko, tiger root and chinen is there.

The above-mentioned effective crude drug used in the apoptosis inhibitor of the second invention of the present application may be used as it is,
You may use these extracts.

Further, the apoptosis inhibitor of the second invention of the present application may be a Chinese herb containing the above crude drug or a composition containing an extract of the above Chinese herb. Specifically, for example, Orengedokuto, Sanohreishinto, Keishibukuryogan, Kaikechinkaito, Rokumimaru, Gyoreito, Hofutsushosan, Saibokuto, Maobuto, Maoubushisaishin. It is hot water and Aoichin Goreisan.

The apoptosis inhibitor of the third invention of the present application is
Oren-gedoku-to, San-horeishin-to, Keishibukuryogan, Keishikashakuyakudaioto, Daijokito, Kakichinkaito, Rokumimaru, Kiboito, Asagoinmaru, Gyoreito, Toki Kenchu-yu, Kawakyucha-sansan, Keishi-ka-ryu-botsu-yu, Kure-yutsu-yu, Hofutsu-sho-san, Tokoku-joki-to, bruise, Daio-botan-sai, Saiboku-to, Mao-to, Mao-zuke It is one kind of herbal medicine selected from the group consisting of Kosaishinto and Aoichingoreisan or its extract.

The blending ratio of the herbal medicine used in the present invention is
In general: (1) Orengedokuto, oreng 1.5-2, Huangshia 1.5-3, Huanggon 3.0, Yamajiko 2-3

(2) Sanohakushinto Daihuang 1-2 Huanggon 1-1.5 Huangren 1-1.5

(3) Keishibukuryomaru Keibushio 4.0 Keibukuryo 4.0 Peony skin 4.0 Momojin 4.0 Shakuyaku 4.0

(4) Keishikashakuyakudaioto Keiryu 4.0 Keishiyaku 6.0 Otsumeu 4.0 Ginger 3-4 (dry ginger 1-2) Licorice 2.0 Daio 1-2

(5) Daishokito Koboku 5.0g Gourmet 3.0g Daihoku 2.0g Anhydrous mirabilite 1.3g

(6) Aoi-chin Kakiyu Aoi-chin Kaki 4-6 Yamajiko 2-3 Daiki 0.8-2

[0025]

(8) Kiboiyu gypsum 10.0 g Boho 4.0 g cinnamon bar 3.0 g Carrot 3.0 g

(9) Asahi-jinmaru Asahi-jin 4-5 Peony medicinal 2.0 Gourmet 2.0 Hoboku 2.0 Daihuang 3.5-4 Apricot kernel 2-2.5

(10) Gyorei-to Soso 2.5-3 Koboku 2.5-3 Chen skin 2.5-3 Boar 2.5-3 Sawarei 2.5-3 Shakuyaku 2.5-3 Shirayu 2. 5-3 Fukuryo 2.5-3 Katsura 2-2.5 Oju 1.5-2 Dry ginger 1.5-2 Licorice 1-2 Shrinking sand 2.0 Yellow Ren 2.0 Peony, shrinking sand, yellow Possible even if there is no series

(11) Tokikenchutou Toki 4.0 Keie 4.0 Ginger 4.0 Otsume 4.0 Peony 5-6 Licorice 2.0 Glue 20.0 (may be omitted)

(12) Kawakyu Tea Conditioning Dispersion 2.0 Kyoukatsu 2.0 Scabbard 2.0 Windbreak 2.0 Thin leaf 2.0 2.0 Licorice 1.5 Tea leaf 1.5 Kawakyu 3.0 Kabushi 4 .0

(13) Keishika Ryukyo Oofu-tou Keishi 3-4 Peony 3-4 Ojumu 3-4 Ginger 3-4 Licorice 2.0 Keel 2.0 2.0 Oyster 3.0

(14) Gourd pork Gourd 3-4 Ginseng 2-3 Ojumu 3-4 Ginger 4-6

(15) Windbreak Tsushosan Toki 1.2 Peony medicine 1.2 River cucumber 1.2 Yamajiko 1.2 Consecutive 1.2 Thin leaflets 1.2 Ginger 1.2 Peony root 1.2 Windbreak 1.2 Mahuang 1.2 Daihuang 1.2 Glauber's salt 1.5 White oak 2.0 Kikyo 2.0 Yellow yellow rice 2.0 Licorice 2.0 Gypsum 2-3 Talc 3-5

(16) Tokokujokito Momojin 5.0 Keishi 4.0 Daihoku 1-3 Glauber's salt 1-2 Licorice 1.5

(17) Healing and bruising Kawakyu 3.0 3.0 Sox 3.0 River bone 3.0 Katsura 3.0 Licorice 1.5 Chorus 1-1.5 Daihoku 1-1.5

(18) Daio Botan Skin Yu Daio 1-2 Botan skin 4.0 Momojin 4.0 Glauber's salt 4.0 Winter melon 4-6

(19) Saiboku-to Saiko 4-7 Half-summer 5-6 Ginger 3.0 Yellow ginseng 3.0 Otsume 2-3 Ginseng 2.0 Licorice 2.0 Borei 5.0 5.0 Koboku 3. 0 Shiso leaf 2.0

(20) Maoyu Mao 4.0-5.0 Apricot kernel 4.0-5.0 Katsura 3.0-4.0 Licorice 1.5-2.0

(21) Mao-fuzi hot-spring hot water Mao-4.0g Hot-spicy 3.0g Shuji-fuzi powder 1.0g

(22) Aoi-chin Gorei-san Sawarei 4.5-6 茯苓 3-4.5 Boar 3-4.5 Katsura 2-3 Aoi chin 3-4

The above-mentioned effective Chinese herb medicine may be used as it is, or an extract thereof may be used. Extracts of crude or herbal medicines include, for example, water, ethanol,
Extracts with various solvents such as acetone, ether or mixtures thereof are preferred, with water extracts being particularly preferred. As a specific extraction method, a crude drug or a herbal medicine is extracted with 8 to 20 times the amount of hot water,
The method of filtering the obtained extract is mentioned. The extract may be dried, if necessary, to give a dry powder.

The apoptosis-inhibiting agents of the first to third inventions of the present application can be administered in any of oral dosage forms and parenteral dosage forms such as injections and infusions.

The apoptosis-inhibiting agents of the first to third inventions of the present application are prepared by mixing the active compound, the effective herbal medicine and the effective Chinese herb medicine with a pharmaceutically acceptable carrier appropriately selected according to the selected dosage form. It can be prepared.

In the case of oral agents, examples of pharmaceutically acceptable carriers include starch, lactose, sucrose, mannitol,
Carboxymethyl cellulose, corn starch, inorganic salts and the like are used. Further, in preparing the oral preparation, a binder, a disintegrating agent, a surfactant, a lubricant, a fluidity promoter, a corrigent, a coloring agent, a fragrance and the like can be further added.
Specific examples thereof include the following.

(Binder) Starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch,
Methylcellulose, sodium carboxymethylcellulose, hydroxypropylcelucose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, macrogol.

(Disintegrant) Starch, hydroxypropyl starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, carboxymethyl cellulose, low-substituted hydroxypropyl cellulose.

(Surfactant) sodium lauryl sulfate,
Soy lecithin, sucrose fatty acid ester, polysorbate 80-1 (lubricant) talc, wax, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol.

(Flowability Accelerator) Light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate.

Examples of oral liquid preparations include suspensions, emulsions, syrups and elixirs. A flavoring agent, a flavoring agent, and a coloring agent may be added to these various dosage forms.

On the other hand, a parenteral preparation can be prepared by dissolving or suspending the active compound of the present invention and the extracts of the effective herbal medicine and the herbal medicine in a solvent according to a conventional method. As the solvent, for example, distilled water for injection,
Physiological saline. Glucose solution, vegetable oil for injection, sesame oil,
Peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol and the like can be used. Further, if necessary, a bactericidal agent, a preservative, a stabilizer, an isotonicity agent, a soothing agent and the like can be added.

The apoptosis-suppressing agents of the first to third inventions of the present application are useful for treating diseases caused by the promotion of apoptosis, such as liver diseases such as hepatitis and diseases caused by deterioration of immune function during stress. In addition, in patients such as Alzheimer's disease, to prevent neuron loss, HIV
It can be used for preventing death of T cells not infected with A. by apoptosis and for preventing cell death of normal cells that are easily damaged by side effects such as anticancer agents.

[0052]

[Examples] Various pharmacological effect tests conducted for confirming the effects of the present invention will be described below.

Test Example 1 The inhibitory effect of TGFβ-induced apoptosis on the rat well-differentiated hepatoma cell line McA-RH8994 was examined for various herbal medicine extracts and commercially available Kampo preparations. In addition,
TGFβ is known to be a major factor in hepatocyte injury and hepatocyte death.

McA-RH8994 cells were treated with Dulbecco's modified medium (DME) supplemented with 10% fetal bovine serum (FBS).
M) at a cell density of 200,000 cells / ml,
[3H] thymidine was added to a final concentration of 0.5 μCi / ml, and the mixture was spread on a 96-well plate. After culturing for 16 hours, the water extract of the crude drug shown in Table 1 and the water-extracted liquid of the Kampo preparation [made by Tsumura Co., Ltd.] shown in Table 2 reach the final concentration (μg / ml) shown in Table 1 and Table 2. So added. One hour later, human recombinant TGFβ1 (King brewing) was added at 0.5 ng / ml. After culturing for another 36 hours, 1/10 volume of 200 mM EDT
A, 5% TrironX-100, 50 mM Tris buffer (pH 8) was added and stirred. After that, stack the glass filter and DEAE filter on top of each other and set them in the harvester, and pass the culture solution through the harvester.
Fragmented DNA was collected on a DEAE filter and non-fragmented DNA was collected on a glass filter. Count on each filter by β scintillation counter, amount of fragmented DNA / {fragmented DN
A amount + unfragmented DNA amount} x 100 (%) is D
It was determined as the NA fragmentation rate.

By the above operation, one sample was not added with TGFβ1 (-TGF), and one sample was added with TGFβ1 one hour later (+ TGF)
Two types of tests were performed. The results are shown in Tables 1 and 2. Tables 1 and 2 show, as controls, those whose DNA fragmentation rates were measured according to the same procedure except that the sample was not added.

[0056]

[Table 1]

[0057]

[Table 2]

As is clear from Tables 1 and 2, in the control, the DNA fragmentation was 32.4% when TGFβ1 was not added, whereas it was 69.3% when TGFβ1 was added. Increased.

On the other hand, DNA induced by the addition of TGFβ1 was observed in all herbal medicines and Chinese herbs.
Increased fragmentation was suppressed. As a result, it was confirmed that the above crude drugs and Chinese herbs could suppress TGFβ1-induced apoptosis in rat hepatoma cell line McA-RH8994.

Test Example 2 Rat normal liver parenchymal cells (primary cultured hepatocytes) prepared by the collagen perfusion method were dispersed in DMEM medium containing 10% FBS at a cell density of 150,000 cells / ml, and the cells were plated on a 96-well plate. It was After culturing for 16 hours, a crude drug water extract shown in Table 3 and a Chinese herb preparation shown in Table 4 [(Co.)
Tsumura] was added to the final concentration (μg / ml) shown in Tables 3 and 4. One hour later, human recombinant TGFβ1 (King brewing) was added at 0.5 ng / ml. After further culturing for 48 hours, the medium was exchanged, and the MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) aqueous solution was added at a final concentration of 1 mg / m 2.
1 was added. After culturing for 4 hours, 100 μl of 10% sodium dodecyl sulfate / 0.01N hydrochloric acid was added,
After incubation for 16 hours, 560 n of culture solution
The absorbance at m was measured, and the number of viable cells was calculated from the calibration curve. The dead cell rate (%) was calculated by the following formula. Dead cell rate (%) = [{number of surviving control cells}-
{Number of viable cells in sample well} / [Number of viable cells in control] × 100 The results are shown in Tables 3 and 4. In addition, Table 3 and Table 4 show the measured dead cell rate according to the same procedure except that the sample was not added.

[0061]

[Table 3]

[0062]

[Table 4]

As is clear from Tables 3 and 4, the dead cell rate in the case of the control was 23.8%. On the other hand, for all herbal medicines and Chinese herbs, T
The dead cell rate in the case of adding GFβ1 was lower than that in the case of the control. As a result, it was confirmed that the above crude drugs and Chinese herbs can suppress TGFβ1-induced apoptosis in normal rat liver parenchymal cells.

Test Example 3 Next, various concentrations were changed for the compound derived from Amaranthus chinensis (Inchinko), capillaryin, 7-methylcapillaryin, 6,7-dimethylesculetin, alcapillin, capyrin, capylene, and capillarin. Then, the inhibitory effect of TGF-induced apoptosis on normal rat hepatocytes (primary cultured hepatocytes) and hepatoma cell line McA-RH8994 was tested according to the procedure described in Test Examples 1 and 2.

Table 5 shows the maximum effective concentration and the DNA fragmentation and dead cell rate at this concentration. In addition,
This result was evaluated as a ratio when the DNA fragmentation by the control and the dead cell rate were 100%.

[0066]

[Table 5]

As is clear from these results, it was found that the compound derived from Anoecium chinensis has an action of suppressing TGFβ1-induced apoptosis.

As described above, the herbal medicines, herbal medicines and compounds shown in Tables 1 to 5 suppress TGF-induced hepatocyte apoptosis, and are effective in treating liver diseases such as viral hepatitis and cirrhosis. There is expected.

Test Example 4 Next, the results of examining the inhibitory effect on adrenocortical hormone-induced apoptosis of various compounds, crude drugs and Chinese herbs will be described.

It is speculated that the decrease in immune function during stress is caused, in part, by atrophy of the thymus and lymphoid tissues due to increased secretion of adrenocortical hormones. The atrophy of thymocytes at that time is presumed to be due to apoptosis induced by adrenocortical hormone (glucocorticoid).

Therefore, the inhibitory activity of apoptosis induced by dexamethasone, which is a synthetic glucocorticoid, was examined in various thyroid medicines, herbal medicines and compounds contained in the herbal medicines using mouse thymocytes.

First, thymocytes were prepared from mouse C3H / HeN and added to RPM1640 medium supplemented with 10% FBS to give 6 cells.
The cells were dispersed at a cell concentration of × 10 ⁶ cells / ml, and this medium was spread on a 96-well plate.

The hot-water extract of crude drug shown in Table 6, the Chinese herb drenched liquid, and the compounds contained in the crude drug were added to the concentrations shown in Table 6 and incubated for 3 hours.
Dexamethasone was added at a concentration of 10 ^-7 M.

After culturing for 15 to 24 hours after adding the sample, 1
/ 10 volume of 200 mM EDTA, 5% TrironX
-100, 50 mM Tris buffer (pH 8) was added,
It was stirred. Then, it was centrifuged at 670 g for 30 minutes to 16 hours to obtain a precipitate. 100 μl of 0.4 g / l for this precipitate
DABA (3,5-diaminobenzoic acid dihydrochloride) was added and reacted at 55 ° C. for 2 hours. Excitation wavelength 407 nm,
Non-fragmented DNA based on a calibration curve measured with a DNA standard solution by measuring fluorescence at a measurement wavelength of 500 nm
The amount was calculated. The amount of fragmented DNA was obtained by subtracting the amount of unfragmented DNA from the total amount of DNA at the time of cell seeding. The DNA fragmentation rate is [{total DNA at cell seeding
Amount}-{amount of unfragmented DNA}] / [total DNA amount at the time of cell seeding] × 100.

By the above-mentioned operation, one sample had 2 dexamethasone not added (-) and dexamethasone added 3 hours after the sample was added (+).
Two types of tests were conducted. The results are shown in Table 6.
Table 6 shows the DNA fragmentation ratio measured according to the same procedure except that the sample was not added, as a control.

[0076]

[Table 6]

As is clear from Table 6, in the control, the DNA fragmentation in the case where dexamethasone was not added was 2
3.6%, and when dexamethasone was added,
It increased to 78.8%.

On the other hand, when the crude drug shown in Table 6, the Chinese herbal medicine and the compound contained in the crude drug were added, the DNA fragmentation rate induced by dexamethasone was lower than that of the control. It was As a result,
The herbal medicines, herbal medicines and compounds contained in herbal medicines shown in Table 6 have an action of suppressing apoptosis induced by dexamethasone, improve immune function decline under stress, and exert immunomodulatory action in various pathological conditions. Expected to do.

An acute toxicity test of the compound which is the active ingredient of the apoptosis inhibitor of the present invention was carried out using male ICR mice.
There were no deaths after oral administration of kg. Therefore, it was confirmed that the compound, which is the active ingredient of the apoptosis inhibitor of the present invention, is highly safe.

Further, each effective crude drug used in the present invention has a long history as a raw material of Kampo medicine, and its safety has been confirmed, and it can be used without fear of side effects. For example, yellow orchard is extremely safe, as evidenced by the fact that no death was observed in mice and rats after oral administration of 15 g / kg, which is the dose limit.

Further, the Kampo medicine used as the apoptosis inhibitor of the present invention has already a long history in Kampo therapy and its safety has been confirmed, and it can be used without fear of side effects. For example, it is clear from the fact that Saibokuto does not cause death in mice and rats after oral administration of 15 g / kg, which is the dose limit,
It is extremely safe.

Formulation examples of an apoptosis inhibitor using the active compound of the present invention will be given below.

Formulation Example 1 (1) Corn starch 44 g (2) Crystalline cellulose 40 g (3) Carboxymethyl cellulose calcium 5 g (4) Light anhydrous silicic acid 0.5 g (5) Magnesium stearate 0.5 g ( 6) Palmatine 10 g Total 100 g (1) to (6) were uniformly mixed according to the above formulation, and compression-molded with a tableting machine to give tablets of 200 mg each.

Each tablet contains 20 mg of palmatin, and 3 to 10 tablets for adults are to be taken in several divided doses per day.

[Formulation Example 2] (1) Crystalline cellulose 84.5 g (2) Magnesium stearate 0.5 g (3) Carboxymethyl cellulose calcium 5 g (4) Berberine 10 g Total 100 g According to the above formulation (1) , (4) and (2) were uniformly mixed, compression-molded, pulverized, added with the remaining amount of (3) and (2), mixed, and compression-molded with a tableting machine. One 200 mg tablet was obtained.

Each tablet contains 20 mg of berberine, and 3 to 10 tablets for adults are to be taken in several divided doses per day.

[Formulation Example 3] (1) Crystalline cellulose 79.5 g (2) 10% hydroxypropyl cellulose ethanol solution 50 g (3) Carboxymethyl cellulose calcium 5 g (4) Magnesium stearate 0.5 g (5) Palmatine 10 g Total 145 g According to the above formulation, (1), (2) and (5) were uniformly mixed, kneaded by a conventional method, granulated by an extrusion granulator, dried and crushed, (3) and (4) were mixed and compression-molded with a tableting machine to obtain 200 mg tablets.

One tablet of this tablet contains 20 mg of palmatine, and 3 to 10 tablets for adults are to be taken in several divided doses per day.

[Formulation Example 4] (1) Corn starch 84 g (2) Magnesium stearate 0.5 g (3) Carboxymethyl cellulose calcium 5 g (4) Light anhydrous silicic acid 0.5 g (5) Capillary 10 g Total 100 g (1) to (5) were uniformly mixed according to the above formulation, compression-molded by a compression molding machine, crushed by a crusher, and sieved to obtain a granule.

1 g of this granule contains 10 g of capillarisin.
It contains 0 mg, and 0.6 to 2 g for adults is to be taken in several divided doses.

[Formulation Example 5] (1) Crystalline cellulose 86.5 g (2) 10% Hydroxypropyl cellulose ethanol solution 35 g (3) 7-Methylcapillarycin 10 g Total 131.5 g According to the above formulation, (1) to (3) was evenly mixed, and the mixture was kneaded. After granulation by an extrusion granulator, the granules were dried and sieved to obtain granules.

1 g of this granule contains 100 mg of 7-methylcapillarycin, and the adult daily dose of 0.6-
Take 2g in several divided doses.

[Formulation Example 6] (1) Corn starch 89.5 g (2) Light anhydrous silicic acid 0.5 g (3) Alcapillin 10 g Total 100 g (1) to (3) were uniformly mixed according to the above formulation. Two
00 mg was filled into a No. 2 capsule.

One capsule of this capsule contains 20 mg of alcapillin, and 3 to 10 capsules per day for adults are to be taken in several divided doses.

[Formulation Example 7] (1) Distilled water for injection 89.5 g (2) Soybean oil 5 g (3) Soybean phospholipid 2.5 g (4) Glycerin 2 g (5) Capillarin 1 g Total amount 100 g Above According to the prescription of (5), (5) was dissolved in (2) and (3), and the solutions of (1) and (4) were added thereto and emulsified to obtain an injection.

[Formulation Example 8] (1) Distilled water for injection Appropriate amount (2) Glucose 200 mg (3) Palmatine 10 g Total amount 15 ml (1) (2) and (3) were dissolved in distilled water for injection Then, the mixture was poured into a 5 ml ampoule and autoclaved at 121 ° C. for 15 minutes to obtain an injection.

Next, examples of the production and preparation of an apoptosis inhibitor using the effective crude drug of the present invention will be given. [Preparation Example 9] Production of orchid extract 2.5 g of orchid was added to 2.5 g of orchid and brewed to a half amount, and the crude drug residue was removed by filtration.
A filtrate was obtained. After cooling the filtrate to room temperature, 7,000 r
Centrifugation was performed at pm for 30 minutes, and the obtained supernatant was freeze-dried to obtain a hot water extract.

[Preparation Example 10] Production of an extract of yellow oak: 100 kg of purified water was added to 10 kg of an oak, and the temperature was 100 ° C.
For 1 hour. The obtained extract was filtered and then spray-dried to obtain 1500 g of dry extract powder.

[Formulation Example 11] Production of Soybean tree extract To 30 kg of Soybean tree, 360 liters of purified water was added, and the mixture was heated to 100 ° C.
For 60 minutes. The obtained extract was subjected to solid-liquid separation by centrifugation, and the obtained separated liquid was spray-dried at 150 ° C to obtain a dry extract powder.

[Preparation Example 12] Production of Red Ginger Yu extract 10 kg of Red Ginger Yu was added to 100 liters of purified water and heated to 100 ° C for 1 hour. The obtained extract is subjected to centrifugation to separate the residue to obtain 80 liters of solution.

20 l of this solution is aseptically clarified by filtration through a 0.3 μm membrane filter (manufactured by Toyo Roshi Kaisha, Ltd.). The obtained filtrate was used as a diafilter G-10T (manufactured by Bioengineering Co .; molecular weight cut off 10,000).
Ultrafiltration with. This ultrafiltration has an internal volume of 2.0
A membrane having a diameter of 152 mm was set on the lower surface of a liter container, the pressure was 3 kg / cm ² , and about 2 liters of purified water was added as the liquid in the container was concentrated.
As a result, 20 liters of ultrafiltrate was obtained.

[Preparation Example 13] Production of cherry bark extract To 50 g of cherry bark, 5 liters of purified water was added, and the mixture was brewed to about half the amount, and the crude drug residue was removed by filtration to obtain a filtrate. The filtrate was cooled to room temperature and then centrifuged at 7,000 rpm for 30 minutes, and the obtained supernatant was freeze-dried to obtain a hot water extract.

[Preparation Example 14] Production of extract of citrus juice 100 kg of purified water was added to 10 kg of citrus juice to give 100
It was extracted by heating at 0 ° C for 1 hour. The obtained extract was filtered and then spray-dried to obtain 1500 g of dry extract powder.

[Formulation Example 15] Production of the extract described above To 30 kg of description, 360 liters of purified water was added, and the mixture was heated to 100 ° C.
For 60 minutes. The obtained extract was subjected to solid-liquid separation by centrifugation, and the obtained separated liquid was spray-dried at 150 ° C to obtain a dry extract powder.

[Preparation Example 16] Production of horse mackerel extract 10 kg horse mackerel was added with 100 liters of purified water and heated to 100 ° C for 1 hour for extraction. The obtained extract was centrifuged and the residue was separated to obtain 80 liters of solution.

20 liters of this solution is aseptically clarified by filtration through a 0.3 μm membrane filter (manufactured by Toyo Roshi Kaisha, Ltd.). The obtained filtrate was used as a diafilter G-10T (manufactured by Bio Engineering Co., Ltd .; molecular weight cutoff of 10,000).
Ultrafiltration with. This ultrafiltration has an internal volume of 2.0
A membrane having a diameter of 152 mm was set on the lower surface of a liter container, the pressure was 3 kg / cm ² , and about 2 liters of purified water was added as the liquid in the container was concentrated.
As a result, 20 liters of ultrafiltrate was obtained.

Formulation Example 17 Preparation of Soybean Granules 1000 g of dry extract powder of Soybean obtained in Example 16
Lactose 980 g and magnesium stearate 20 g
This mixture was mixed with a single-shot tableting machine, and a slug tablet having a diameter of 20 mm and a weight of 2.3 g was crushed with an oscillator, sized and sieved to give a granule having a particle size of 20 to 50 mesh. Got

[Preparation Example 18] Preparation of yellow oak tablets 250 g of dried oak powder obtained in Example 12 was mixed with 47 g of crystalline cellulose and 3 g of magnesium stearate, and the mixture was tabletted with a single-shot tableting machine. Then
A tablet having a diameter of 9 mm and a weight of 300 mg was produced. One tablet of the present invention contains 250 mg of dried yellow oak powder.

[Formulation Example 19] Preparation of yellow orchid capsules Hard capsules were filled with 500 mg of yellow orchid dry extract powder obtained in Example 9 to prepare capsules.

[Preparation Example 20] Preparation of Red Cucumber Cucumber Injection Preparation 300 g of allantoin (not containing a pyrogen) was dissolved in 20 liters of the red filtrate of Red Cucumber Cucumber obtained in Example 12. This was dispensed into 900 vials and freeze-dried to obtain a powder injection. This injection 1 vial contained 406 mg of the freeze-dried product and was easily dissolved in 10 ml of purified water. In addition, the injection solution after dissolution is 9
It had a transmittance of 2% (550 nm) and complied with the Japanese Pharmacopoeia test method for pyrogenic substances.

[0111] Examples of production and preparation of an apoptosis inhibitor using the Chinese medicine of the present invention will be given below.

[Preparation Example 21] Production of an extract of Orengedokuto 17g of Orengedokuto (17 g, 4.0 g of Oren, 3.0 g of Kashiwa, 6.0 g of Huanggo and 4.0 g of Yamaboshi) was purified. Water was added and brewing was performed until the amount became almost half (30 to 40 minutes), and the crude drug residue was removed by filtration to obtain a filtrate. After cooling the filtrate to room temperature, centrifugation was performed at 7,000 rpm for 30 minutes, and the obtained supernatant was freeze-dried to obtain a hot water extract.

[Preparation Example 22] Production of Keishikaryukotsu Oyoketo Keishikaryukotsu Oyoketo (3 parts Keishika, 3 parts peony root, 3 parts daikon, ginger 3 parts, licorice 2 parts, keel 2 parts, oyster) 0.3 liter) of prescription crude drug (10 kg) was added with 120 liters of purified water, and the mixture was heated and extracted at 100 ° C. for 1 hour. The obtained extract was filtered and then spray-dried to obtain a dry extract powder.

[Preparation Example 23] Production of extract of fungus-chin-gorei-san Fungus-chin-gorei-san (5 parts Sawarei, 3 parts Furei, 3 parts boar, 3 shaku)
Part, katsura 2 parts, chinenshiki 3 parts) to 30 kg of a prescription crude drug was added 360 liters of purified water and extracted at 100 ° C. for 60 minutes. The obtained extract was subjected to solid-liquid separation by centrifugation, and the obtained separated liquid was spray-dried at 150 ° C to obtain a dry extract powder.

[Preparation Example 24] Production of an extract of Saiboku-to Saiboku-to (4 parts of saiko, 6 parts of half-summer, 3 parts of ginger, 3 parts of yellow rice)
2 parts of large jujube, 2 parts of carrot, 2 parts of licorice, 5 parts of peony, 3 parts of magnolia,
100 liters of purified water was added to 10 kg of a prescription crude drug of Shiso leaf 2 parts), heated, and extracted for 1 hour after reaching 100 ° C. The obtained extract was centrifuged and the residue was separated to obtain 80 liters of solution.

20 liters of this solution is aseptically clarified by filtration through a 0.3 μm membrane filter (manufactured by Toyo Roshi Kaisha, Ltd.). The obtained filtrate was used as a diafilter G-10T (manufactured by Bioengineering Co .; molecular weight cut off 10,000).
Ultrafiltration with. This ultrafiltration has an internal volume of 2.0
A membrane having a diameter of 152 mm was set on the lower surface of a liter container, the pressure was 3 kg / cm ² , and about 2 liters of purified water was added as the liquid in the container was concentrated.
As a result, 20 liters of ultrafiltrate was obtained.

[Formulation Example 25] Production of an extract of Bofutsushosan Sanfufutsushosan 28g (toki 1.2g, peony drug 1.2g, river cucumber 1.2g, yamajuko 1.2g, rendan 1.2g, Thin leaf 1.2g, Ginger 1.2g, Peony root 1.2g, Windbreak 1.2
g, mahuang 1.2g, rhubarb 1.5g, mirabilite 1.5g, white oak 2.0g, bellflower 2.0g, yellow rice 2.0g, licorice 2.0
g, gypsum 2.0 g, talc 3.0 g), 280 ml of purified water was added, and brewing was performed until the amount became almost half (30 to 40 minutes), and the crude drug residue was removed by filtration to obtain a filtrate. After cooling the filtrate to room temperature, centrifugation was performed at 7,000 rpm for 30 minutes, and the obtained supernatant was freeze-dried to obtain a hot water extract.

[Formulation Example 26] Production of Kakuchin-Kanto Hot water of 120-liter purified water was added to 10 kg of a prescription crude drug of Kakuchin-Kanto (4 parts of Kakchin-chan, 2 parts of Yamaboshi, 1 part of Daio).
Heat extraction was performed at 0 ° C. for 1 hour. After filtering the obtained extract,
Spray drying was performed to obtain a dry extract powder.

[Preparation Example 27] Preparation of extract of Sankyoshashinto Sankireishinto (1 part of Daio, 1 part of yellow gourd, 1 part of oren) 30 kg of crude drug was added with 360 liters of purified water to obtain 100 ℃
For 60 minutes. The obtained extract was subjected to solid-liquid separation by centrifugation, and the obtained separated liquid was spray-dried at 150 ° C to obtain a dry extract powder.

[Preparation Example 28] Production of an extract of Kureiyu-to Kurei-yu (4 parts of Kurei-yu, 4 parts of ginger, 3 parts of Otsuki, 3 ginseng)
100 liters of purified water was added to 10 kg of the prescription crude drug of Part 1), heated, and extracted for 1 hour after reaching 100 ° C. The extract obtained is centrifuged to separate the residue and
Get 0 liters.

20 liters of this solution is aseptically clarified by filtration with a 0.3 μm membrane filter (manufactured by Toyo Roshi Kaisha, Ltd.). The obtained filtrate was used as a diafilter G-10T (manufactured by Bio Engineering Co., Ltd .; molecular weight cutoff of 10,000).
Ultrafiltration with. This ultrafiltration has an internal volume of 2.0
A membrane having a diameter of 152 mm was set on the lower surface of a liter container, the pressure was 3 kg / cm ² , and about 2 liters of purified water was added as the liquid in the container was concentrated.
As a result, 20 liters of ultrafiltrate was obtained.

[Formulation Example 29] Production of an extract of Tokikenchutou Tokikenchutou (4 parts of Tokiken, 4 parts of Keishi, 4 parts of ginger, 4 parts of Otsuki)
Part, peony root 5 parts, licorice 2 parts) to 100 kg of purified water was added to 10 kg of prescription crude drug until it became almost half (30-4).
It was brewed for 0 minutes) and the crude drug residue was removed by filtration to obtain a filtrate. After cooling the filtrate to room temperature, 7,000 rpm
Centrifugation was performed for 30 minutes, and the resulting supernatant was freeze-dried to obtain a hot water extract.

[Preparation Example 30] Production of Keishibukuryogan Keibishibukuryogan (Keiobushi 4 parts, bukuryo 4 parts, peony skin 4 parts, peach kernel 4)
Part, peony root 4 parts) was added with 120 liters of purified water and extracted by heating at 100 ° C. for 1 hour. The obtained extract was filtered and then spray-dried to obtain a dry extract powder.

[Preparation Example 31] Production of granules of Aochin Goreisan [0124] 1000 g of dried extract powder of Aoichin Goreisan prepared according to Example 23 was mixed with 980 g of lactose and 20 g of magnesium stearate, and this mixture was spun single-shot. A slug tablet having a diameter of 20 mm and a weight of 2.3 g was crushed with an oscillator, sized and sieved to obtain a granule having a particle size of 20 to 50 mesh.

[Preparation Example 32] Preparation of tablets of Keishika-ryūkoku-boi-to 250g of dried extract powder of Keishi-ka-ryu-boi-to prepared in Example 22 was mixed with 47g of crystalline cellulose and 3g of magnesium stearate, and this mixture was mixed. Was tableted with a single-shot tableting machine to produce tablets having a diameter of 9 mm and a weight of 300 mg. One tablet of the present invention contains 250 mg of a dry extract powder of Keishikaryuku Oyoketo.

[Formulation Example 33] Preparation of capsules Hard capsules were filled with 500 mg of the dry extract powder of Oren-gedokuto prepared in Example 21, to prepare capsules.

[Formulation Example 34] 300 g of allantoin (free of heat generating substance) was added to and dissolved in 20 liters of the ultrafiltrate of Saibokuto obtained in Example 24. This was dispensed into 900 vials and freeze-dried to obtain a powder injection.
One vial of this injection contained 406 mg of the freeze-dried product and was easily dissolved in 10 ml of purified water. Further, the injection solution after dissolution had a transmittance of 92% (550 nm), and was suitable for the pyrogenic substance test method of the Japanese Pharmacopoeia.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location A61K 35/78 A61K 35/78 T ACS ACSK 31/01 31/01 31/32 31/32 31 / 435 31/435 35/84 35/84 AZ // C07D 311/16 101 C07D 311/16 101 455/03 455/03 (72) Inventor Kazuo Ogawa 3586 Yoshihara, Ami-cho, Inashiki-gun, Ibaraki Tsumura Co., Ltd. (72) Inventor Kazunori Fukuda 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki Tsumura Co., Ltd.

Claims (3)

[Claims] 1. Apoptosis containing at least one selected from the group consisting of berberine, palmatine, capillarisin, 7-methylcapillarisin, 6,7-dimethylesculetin, alcapillin, capyrin, capylene and capillarin as an active ingredient. Inhibitor. 2. Yellow ore, yellow oak, Suoki, red Qin cucumber, cherry bark,
Inhibition of apoptosis containing at least one herbal medicine selected from the group consisting of 樸 荟, 虸, maranko, roadside, shrub, makigashi, mao, 苯 苓, frankincense, maeko, tiger root and chinen Agent. 3. Orengedokuto, Sanohreishinto, Keishibukuryogan,
Keishikashakuyakudaioto, Daijokito, Aoichinkakito, Rokumimaru, Kiboito, Asagojinmaru, Gyoreito, Tokikenchuyu, Kawakyucha Osamu, Keishikaryukotsu Oyster Selected from the group consisting of hot water, gojutsuyu, hofutsushosan, tokokujokito, bruise, daio-botan-sai, saiboku-to, mao-to, mao-bushisaishin-to, and chichin-gorei-san. An apoptosis inhibitor containing one Chinese herb medicine.