JPH09241166A - Synthesis suppressant containing ginsenosides for protein belonging to hsp60 family - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a synthesis suppressant for a heat shock protein, comprising ginsenosides as an active ingredient and useful for treating autoimmune diseases such as insulin-dependent diabetes mellitus or chronic articular rheumatism. SOLUTION: This synthesis suppressant for a heat shock protein comprises ginsenoside Rg1 represented by the formula (G1c is β-D-glucopyranosyl) as an active ingredient in an amount of 0.01-99wt.%, preferably 0.1-80wt.%. The compound represented by the formula has suppressing actions on the expression of a protein, having 57-68kDa molecular weight and belonging to the heat shock protein (HSP60 family). The compound represented by the formula is obtained by carrying out the chemical synthesis or extraction and purification from a natural product Ginseng Radix or Ginseng Radix Rubri, or a commercially available product may be used. The daily dose thereof for an adult is 1mg to 10 g, expressed in terms of the amount of the ginsenoside Rg1 and perorally or parenterally administered.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a group of heat shock proteins containing ginsenosides, particularly ginsenoside Rg ₁ as an active ingredient and having a molecular weight of 57 kilodaltons (kD) to 68 kD (hereinafter referred to as HSP60 family). ) Related to a protein synthesis inhibitor. The synthesis inhibitor of the protein belonging to the HSP60 family according to the present invention is particularly considered to be an autoimmune disease in which the protein belonging to the HSP60 family is involved in the onset by suppressing the intra-tissue synthesis of the protein belonging to the HSP60 family. , For example,
It is possible to effectively improve the physiological condition of patients with diseases such as type I diabetes and rheumatoid arthritis, and to effectively treat autoimmune diseases such as type I diabetes and rheumatoid arthritis.

[0002]

2. Description of the Related Art In recent years, autoimmune diseases have become a major problem. An autoimmune disease is a disease in which the immune system, which normally should not attack the components that make up your body, reacts with your own tissue and destroys it.
Includes type I diabetes and rheumatoid arthritis. For example, in recent years in Japan, with the development of economy, society, and culture, as well as the improvement of living standards and changes in lifestyles, the number of diabetic patients has increased significantly, and the condition has become more severe and complicated. Advances in diabetology have improved patient prognosis, but have increased frequent retinopathies, nephropathy, and neuropathy, as well as accelerated arterial stiffness, which has severely impaired health and social activities . Among diabetes, type I diabetes (insulin-dependent diabetes mellitus; IDD)
The incidence of M) has increased several fold in many countries in the last few decades, with 1% of currently living humans becoming I by the age of 70.
It is expected that type 2 diabetes will occur.

[0003] Type I diabetes is a disease in which only β cells of the pancreatic islets of Langerhans, which are insulin-producing cells, are autoimmunely destroyed, resulting in an insulin deficiency state, and is an organ-specific autoimmune disease (Atkinson, MA et al .:
"Sci. Am.", 263 : 42-49, 1990; Todd JA .: "Immunol.
Today ", 11 : 122-129, 1990). The autoimmune process that causes type I diabetes is very strictly limited to the pancreas,
Often develops before becoming an adult. When type I diabetes clinically develops, there is inflammation of the islets (isletitis), and most of the β cells that produce insulin are specifically lost (Atkinson, MA, et al .: "Sci. A
m. ", 263 : 62-67, 1990). The clinical manifestation of diabetes only appears after the majority of β-cells (probably 90% or more) have been destroyed to the extent that they cannot regenerate, and the survival of the patient is out of insulin. The autoimmune response is already irreversibly damaged by the time it can be detected by clinical diagnosis, and many do not show significant subjective symptoms, etc. Type I diabetes has many problems.

[0004] Rheumatoid arthritis is a chronic inflammatory disease in which the synovium of the joint is the main lesion. The site of the lesion is sometimes not limited to the synovium of the joint, and it is not uncommon for it to extend to the whole body. The inflammation that initially occurs in the synovium of the joint eventually causes synovial proliferation, further destruction of cartilage and bone, and destruction of joint tissue. As a result, the patient is not only severely restricted socially and at home, but also has a considerable financial burden. The number of patients with rheumatoid arthritis accounts for 0.1-0.3% of the population. This is an example of confirming rheumatoid arthritis, and it is expected that the number of patients will increase to about 10 times that of rheumatoid arthritis, including suspected cases and rheumatoid arthritis-related diseases.

On the other hand, heat shock proteins (heat shock proteins)
protein; also called HSP, stress protein)
Cells with some stress, such as heat, heavy metals, drugs,
A group of proteins expressed in cells by stimulation with amino acid analogs or hypoxia (low oxygen concentration). Heat shock proteins are ubiquitous in nature and are produced by higher animals, including bacteria, yeast, plants, insects, and humans.

[0006] HSPs are of various types,
Due to the molecular weight, the HSP90 family (for example, 9
0 kD or 110 kD HSP, etc.), HSP70 family (eg, 70-73 kD HSP, etc.), HS
P60 family (eg, 57-68 kD HSP etc.), small molecule HSP family (eg, 20 kD, 2
5-28 kD or 47 kD HSP). In the present specification, an HSP having a specific molecular weight is indicated by an HSP and a numeral described immediately after the HSP. For example, an HSP having a molecular weight of 60 kD is referred to as “HSP60”. As described above, there are many types of HSPs, which differ not only in molecular weight but also in structure, function, or property. In addition to the response to stress, some of these proteins are constitutively synthesized and, under normal circumstances, regulate protein folding, unfolding, protein subunit assembly, and protein membrane trafficking. It has been shown to play an essential physiological role. These functions as heat shock proteins are called molecular chaperones.

[0007] One of the attentions regarding the etiology of autoimmune diseases is molecular homology. In other words, when a self-antigen has a common antigenicity with a foreign antigen such as a microorganism, antibodies and sensitized lymphocytes produced by microbial infection attack their own tissues by cross-reaction, resulting in the development of an autoimmune disease. (Atkinson, MA et al .: "Sci. A
m. ", 263 : 42-49, 1990; Shinha, AA et al .:" Scie
nce ", 248 : 1380-1388, 1990). For example, bacterial HSP
Proteins belonging to 60 families include tuberculosis, leprosy,
It is a major antigen such as syphilis, veterans' disease, or Lyme disease (Young, RA et al .: "Cell", 59 : 5-8, 1989),
In addition, proteins belonging to the HSP60 family of bacteria have strong immunogenicity and have molecular homology with their own (host human) proteins, so that they are not affected by infectious disease-triggered molecular homology. It is believed that autoimmune diseases develop.

For example, an antibody (64 kD) that reacts with a 64 kD protein detected in the blood of diabetic patients and their families
D autoantibodies) are β-cell specific, and often appear immediately before diabetes is diagnosed. That is, pancreatic islet cell antigen, which is considered to be the cause of onset in type I diabetes, is a glycoprotein having a molecular weight of 64 kD (Baekkeskov, S. e.
t al .: "Nature", 298 : 167-169, 1982). 64
Antibodies against the kD protein are not limited to human diabetes (Atkinson, MA et al .: "Lancet", 335 : 1357-136).
0, 1990), BB rats (Baekkeskov, S. et al .: "Scie
nce ", 224 : 1348-1350, 1984) and NOD mice (Atkins
on, MA et al .: "Diabetes", 37 : 1587-1590, 1988)
As described above, it is also detected in a type I diabetes model animal that naturally develops type I diabetes and exhibits many characteristics of human type I diabetes. The 64 kD protein of pancreatic β-cell in type I diabetes may be a heat shock protein because it is induced by cytokines and heat stimulation.

The 64 kD protein of the pancreatic Langerhans islet β-cell in NOD mouse, a model animal of type I diabetes, is derived from H. tuberculosis (Mycobacterium tuberculosis).
It has been shown to be an autoantigen that is immunologically cross-reactive with antibodies to proteins belonging to the SP60 family. As described above, the immunological crossover between the protein belonging to the HSP60 family and the 64 kD protein autoantigen was observed.
The D protein may be a member of the HSP60 family, and it has been suggested that an autoimmune mechanism that crosses an epitope of a protein belonging to the HSP60 family is involved in the development of type I diabetes. When a T lymphocyte clone having specificity for a protein belonging to the HSP60 family of Mycobacterium tuberculosis is transferred, it causes Langerhans insulitis and hyperglycemia in young NOD mice. In addition, when a protein belonging to the HSP60 family of Mycobacterium tuberculosis is injected into NOD mice by an immunogenic administration method, that is, by injection into a NOD mouse together with an adjuvant, diabetes can be developed early (Elias, D. et al .: "Proc. Natl. Acad. . Sci.
USA ", 87 : 1576-1580, 1990). HS of mycobacteria
These facts that the immune response of animals to proteins belonging to the P60 family cause type I diabetes are due to the attack by the immune system on antigens that cross-react with antibodies to proteins belonging to the HSP60 family of mycobacteria, damaging β cells. It is shown that.

It is known that proteins belonging to the HSP60 family are related to rat adjuvant arthritis, which is an animal model for rheumatoid arthritis, and to human rheumatoid arthritis. For example, in the case of rheumatoid arthritis, among the heat shock proteins that are bacterial cell proteins, proteins belonging to the HSP60 family have been found to have molecular homology to proteoglycans present in articular cartilage. The involvement of protein-reactive T lymphocytes belonging to the HSP60 family has been shown in adjuvant arthritis in rats ("C
urr. Top. Microbiol. Immunol. ", 145 : 27-83, 198.
9). The disease is transferred to irradiated immunologically naive rats by transferring a clone of T lymphocytes reactive to proteins belonging to the HSP60 family of M. tuberculosis. ("Science", 219 : 56-5
8, 1983; "Nature", 331 : 171-173, 1988). The T lymphocytes also show cross-reactivity with joint proteoglycans ("Proc. Natl. Acad. Sci. USA", 82 : 5117-512).
0, 1985). Regulatory T lymphocytes induced by proteins belonging to the HSP60 family are also recognized in arthritis due to streptococcus and pristine. Thus, adjuvant arthritis appears to be an autoimmune disease caused by protein T lymphocytes belonging to the anti-HSP60 family. Also, in human juvenile rheumatoid arthritis, HS
Involvement of protein-reactive T lymphocytes belonging to the P60 family is considered.

In addition, T lymphocytes that specifically react with proteins belonging to the HSP60 family derived from mycobacteria have been extracted from the synovial fluid of patients with rheumatoid arthritis ("Lancet", II : 478-). 480, 1988; "Na
ture ", 339 : 226, 1989;" Annu. Rev. Immunol. ", 1
1 : 637, 1993). Thus, the mycobacterial HS
Proteins that show cross-reactivity with proteins belonging to the P60 family are found at high concentrations in the cartilage / pannus junction of rheumatoid arthritis, whereas they are found in normal tissues and tissues that exhibit chronic inflammation due to other diseases. No ("Scand. J. Immunol.", 31 : 283-288, 1990). Furthermore, antibodies against proteins belonging to the HSP60 family are detected in human and rat rheumatoid arthritis (Kaufmann, SHE, et al .: "Immunol. Today", 11 :
129-136, 1990), it is possible that the etiology of rheumatoid arthritis is autoimmunity against self antigens similar in structure to proteins belonging to the HSP60 family of mycobacteria. Thus, the presence of an immune response to proteins belonging to the HSP60 family is associated with both rat and human arthritis.

[0012]

SUMMARY OF THE INVENTION In view of the above circumstances, the present inventors have been able to effectively improve the physiological condition of patients with autoimmune diseases such as type I diabetes and rheumatoid arthritis, In order to develop a method that can effectively treat immune diseases, various studies have been made on compounds that exhibit a synthesis inhibitory effect on proteins belonging to the HSP60 family. As a result, the inventors
Surprisingly, it was discovered that ginsenosides, which are components such as carrots and ginseng, in particular ginsenoside Rg ₁ , specifically inhibit the synthesis of proteins belonging to the HSP60 family in cells of tissues showing pathological conditions. That is, it was found that administration of ginsenosides suppresses intracellular synthesis of proteins belonging to the HSP60 family, and therefore treatment of autoimmune diseases such as type I diabetes and rheumatoid arthritis is possible. is there. The present invention is based on these findings.
An object of the present invention is to provide a synthetic inhibitor of a protein belonging to the HSP60 family, which can effectively treat autoimmune diseases such as type 2 diabetes and rheumatoid arthritis.

[0013]

Therefore, the present invention is characterized by containing ginsenosides, especially ginsenoside Rg ₁ as an active ingredient, and a heat shock protein having a molecular weight of 57 to 68 kilodaltons ( That is, it relates to a synthetic inhibitor of a protein belonging to the HSP60 family). In this specification, "HS
As described above, the "P60 family" has a molecular weight of 57.
It means a group of heat shock proteins of kD to 68 kD.
Examples of proteins belonging to the HSP60 family include HSP60 (that is, a heat shock protein having a molecular weight of 60 kD), HSP58 (that is, a heat shock protein having a molecular weight of 58 kD), HSP65 (that is, a heat shock protein having a molecular weight of 65 kD), or GroEL. (Ie, a prokaryote, for example, a heat shock protein having a molecular weight of about 64 kD such as Escherichia coli).

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. The synthesis inhibitor of the present invention contains ginsenosides as an active ingredient. In the present specification, ginsenosides (ginsenosides; ginsenosides) are, for example, ginsenoside Ro (ginsenoside Ro;
ginsenoside Ro; Chixetsusaponin V;
chikusetsusaponin V; saponin A;
saponin A), ginsenoside Ra ₁ (ginsenosides Ra _1; ginsenoside Ra _1), ginsenoside Ra ₂ (ginsenosides Ra _2; ginse
noside Ra _2), ginsenoside Rb ₁ (ginsenosides Rb _1; ginsenoside Rb _1; saponin D; saponin D), ginsenoside Rb
₂ (ginsenoside Rb ₂ ; ginsenoside R
b ₂ ), ginsenoside Rb ₃ (ginsenoside Rb ₃ ;
ginsenoside Rb ₃ ), ginsenoside R
c (ginsenoside Rc; ginsenoside R
c), ginsenoside Rd (ginsenoside Rd; gin
ginsenoside Rd), ginsenoside Re (ginsenoside Re), ginsenoside Rf (ginsenoside Rf; ginsenos)
ideRf), ginsenoside Rg ₁ (ginsenoside R
g ₁ ; ginsenoside Rg ₁ ), ginsenoside Rg ₂ (ginsenoside Rg ₂ ; ginsenosi)
de Rg ₂ ; Chixetsusaponin I; chikuset
susaponinI), ginsenoside Rg ₃ (ginsenoside Rg _3; ginsenosideRg _3), ginsenoside Rh ₁ (ginsenosides Rh _1; ginse
nosin Rh ₁ ), ginsenoside Rh ₂ (ginsenoside Rh ₂ ; ginsenoside Rh ₂ ),
20-glucodinsenoside Rf (20-glucoginsenoside Rf; 20-glucoginsenoside Rf)
Rf), malonyl ginsenosides Side Rb ₁ (Maroni alginic Seno Cid Rb _1; maronylginsenosi
de Rb ₁ ), malonyl ginsenoside Rb ₂ (malonylginsenoside Rb ₂ ; maronylginsen)
oside Rb ₂ ), malonyl ginsenoside Rc
(Malonylginsenoside Rc; maronylgins
enosideRc), malonylzine senoside Rd
(Malonylginsenoside Rd; maronylgins
enoside Rd), Chixetsusaponin Ia (ch
ikusetsusaponin Ia), chikusetsusaponin Ib
b), Chixetsusaponin III (chikusetsus)
aponin III), Chixetusa saponin IV (chik)
usetsusaponin IV; saponin B; sapo
nin B), Chixetsusaponin IVa (chikuse)
tsusaponin IVa; saponin C; sapon
in C), protopanaxadiol (protopadiol)
taxidiol), protopanaxatriol (pr
otopanaxtriol), oleanolic acid (o
leanonic acid) or the stereoisomers of these compounds. In the present invention, these ginsenosides can be used alone or in combination with a plurality of different ginsenosides.

[0015] As ginsenosides which can be used as active ingredients in the synthesis inhibitor of the present invention, in particular, ginsenoside Rg ₁ being most preferred. Ginsenoside Rg ₁ (ginsenosides Rg _1; ginsenos
ide Rg ₁ ) is of the formula (I):

Embedded image (Wherein, Glc is a β-D-glucopyranosyl group)
And a molecular formula of C ₄₂ H ₇₂ O ₁₄ and a molecular weight of 801.03
And contained in crude drugs such as carrots and kojin.

The ginsenosides contained in the synthesis inhibitor of the present invention can be prepared by chemical synthesis or by extracting and purifying from natural products. Alternatively, a commercially available product may be used. When extracting ginsenosides used as an active ingredient in the synthesis inhibitor of the present invention from natural products, for example, whole or part of a plant containing ginsenosides (for example, whole plant, leaf, root,
The rhizome, stem, root bark, or flower) is used as it is, or is simply processed (for example, dried, cut, blanched, steam-heated, or powdered) (for example, a crude drug). The extraction conditions are not particularly limited as long as they are generally used for plant extraction. Examples of plants containing ginsenosides include, but are not limited to, ginseng, Panax ginseng CA Meyee of the family Araliaceae.
r; Panax schinseng Nees), Panax ginseng [Panaxjaponicus C. et al.
A. Meyer; Panax sinsengNe
es var. japonicum Makino; P
anax pseudo-ginseng (Wil
l. ) Subsp. japonicus Har
a], Panax notoginseng [Panax notogin
Seng (Burkill) F.S. H. Chen; Pa
nax sanchi Hoo; Panaxpseud
o-ginseng Wallich var. not
oginseng (Burkill) Hoo et
Tseng] or American carrot (Panax q
uinquefolium L. ) Etc. can be used.

When ginsenosides in the present invention are extracted from crude drugs, the extraction is not limited thereto, but, for example, it is preferable to extract ginsenosides from carrot or kojin. Carrot (Ginseng Radix) means a root excluding fine roots of Panax ginseng or a lightly blanched root thereof, and these portions can be used alone or in any combination. In addition, kojin (red ginseng; Red Ginseng; Ginseng)
Radix Rubra) means steamed panax ginseng roots, and these portions can be used alone or in any combination.

[0018] carrot extract or red ginseng extract can be used as an active ingredient in the synthesis inhibitor according to the present invention, ginsenosides above, it is sufficient that, especially containing ginsenoside Rg _1, therefore, the carrot or red ginseng crude The extract can be used. As a method for producing a carrot extract or a ginseng extract that can be used in the present invention, a carrot or a ginseng is extracted by water (for example, hot water, preferably hot water) or by extraction with an organic solvent. ,Obtainable. Examples of the organic solvent include alcohols having 1 to 6 carbon atoms (eg, methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol, or butyl alcohol), fatty acid esters (eg, methyl acetate, ethyl acetate, propyl acetate, Or butyl acetate), ketone (eg, acetone or methyl isobutyl ketone), ether, petroleum ether, n
Using hexane, cyclohexane, toluene, benzene, halogen derivatives of hydrocarbons (for example, carbon tetrachloride, dichloromethane or chloroform), pyridine, glycol (for example, propylene glycol or butylene glycol), polyethylene glycol, or acetonitrile; These organic solvents can be used alone or in an appropriate combination, mixed at a certain ratio, and further used in an anhydrous or water-containing state. Preferably, methyl alcohol and the like are desirable. As a method of water extraction or organic solvent extraction, a method used for ordinary crude drug extraction can be used. For example, 3 to 300 parts by weight of water or an organic solvent is used for 1 part by weight of (dried) carrot or kojin. Then, it is desirable to heat and reflux at a temperature lower than the boiling point while stirring, or to perform ultrasonic extraction at room temperature. The extraction step is usually performed for 5 minutes to 7 days, preferably for 10 minutes.
The extraction time can be shortened by carrying out for a period of minutes to 24 hours and, if necessary, adding auxiliary means such as stirring.

After completion of the extraction step, a crude extract can be obtained by separating insolubles from the water or organic solvent extract by a suitable method such as filtration or centrifugation. Incidentally, in the synthesis inhibitors of the present invention, extracted from natural products, fractionated ginsenosides, especially in the case of using ginsenoside Rg _1, without particular purification the crude extract may be used as it is. In addition to a hot water extract or an organic solvent extract by a conventional method, a purified extract obtained by further treating the crude extract with various organic solvents or adsorbents is also used as an active ingredient of the synthesis inhibitor of the present invention. be able to. A carrot extract or a ginseng extract containing these crude extracts and purified extracts after various purification treatments may be used as-extracted solution or an extract obtained by concentrating the solvent, or may be used as a solvent. , A powder obtained by distilling off and drying, further a crystallized and purified substance, or a viscous substance may be used, or a diluent thereof may be used. The carrot extract or ginseng extract thus obtained contains ginsenosides contained in the ginseng or ginseng as a mixture, and at the same time contains impurities derived from the carrot or ginseng as the raw material.

The synthetic inhibitor of the present invention comprises ginsenosides or an extract of a plant containing ginsenosides, for example, an extract of a crude drug containing ginsenosides (particularly, a carrot extract or a ginseng extract). It can be administered to animals, preferably mammals (especially humans), alone or preferably together with conventional pharmaceutically or veterinarily acceptable carriers. The administration dosage form is not particularly limited, for example, powders, fine granules,
Granules, tablets, capsules, suspensions, emulsions,
Examples include oral preparations such as syrups, extracts and pills, and parenteral preparations such as injections, external solutions, ointments, suppositories, creams for topical administration, and eye drops. These oral preparations include, for example, gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soy lecithin, sucrose, fatty acid esters, talc, stearic acid. Excipients such as magnesium, polyethylene glycol, magnesium silicate, anhydrous silicic acid, or synthetic aluminum silicate, binders, disintegrants, surfactants, lubricants, glidants, diluents, preservatives, coloring It can be produced according to a conventional method using an agent, a flavor, a flavoring agent, a stabilizer, a humectant, a preservative, an antioxidant, or the like. For example,
Capsules prepared by mixing and filling ₁ part by weight of ginsenoside Rg1 and 99 parts by weight of lactose.

Examples of parenteral administration methods include injection (subcutaneous, intravenous, etc.) and rectal administration. Of these, injections are most preferably used. For example, in the preparation of an injection, ginsenosides (particularly ginsenoside Rg ₁ ) as an active ingredient, or extracts of plants containing ginsenosides, for example, extracts of crude drugs containing ginsenosides (particularly carrots) Extract or kojin extract), in addition, for example, water-soluble solvents such as physiological saline or Ringer's solution, water-insoluble solvents such as vegetable oils or fatty acid esters, isotonic agents such as glucose or sodium chloride, solubilizing agents, A stabilizer, preservative, suspending agent, emulsifier, or the like can be optionally used. Further, the synthetic inhibitor of the present invention may be administered using a sustained-release preparation technique using a sustained-release polymer or the like. For example,
The synthetic inhibitors of the present invention can be incorporated into a pellet of ethylene vinyl acetate polymer and the pellet can be surgically implanted into the tissue to be treated.

The synthesis inhibitor of the present invention is not limited to this.
%, Preferably 0.1 to 80% by weight. In addition, extracts of plants containing ginsenosides, for example, extracts of crude drugs containing ginsenosides (particularly, carrot extracts or kojin extract)
Can be prepared by appropriately adjusting the amount of ginsenosides (particularly, ginsenoside Rg ₁ ) contained therein to the above-mentioned range. In addition, a plant extract containing ginsenosides, for example, a synthetic inhibitor containing an extract of a crude drug containing ginsenosides (particularly, a carrot extract or a ginseng extract) as an active ingredient, a formulation for oral administration. In such a case, it is preferable to formulate a preparation using a pharmaceutically acceptable carrier.

The dose of the synthetic inhibitor of the present invention depends on the type of disease, the age, sex, weight, degree of symptoms and the method of administration of the patient, and is not particularly limited, and the amount of ginsenoside Rg ₁ is not particularly limited. As usual 1 per adult
About 10 mg to 10 g is orally or parenterally administered in about 1 to 4 times a day. Furthermore, the use is not limited to pharmaceuticals, and various uses, for example, functional foods and health foods can be given in the form of food and drink.

[0024]

[Action] As described above, ginsenosides contained in synthesis inhibitors of the present invention, particularly ginsenoside Rg _1,
Since it has an action of specifically suppressing the synthesis of proteins belonging to the HSP60 family in cells, administration of the ginsenosides specifically reduces the biosynthesis of proteins belonging to the HSP60 family in cells. Therefore,
The ginsenosides can be used for prevention and treatment of autoimmune diseases associated with the onset of proteins belonging to the HSP60 family, such as type I diabetes and rheumatoid arthritis.

[0025]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention. Example 1: Measurement of HSP expression level in human cultured cancer cells (1) Culture of human cultured cancer cells Human lung cancer cell line H69 (ATCC HTB 119)
RPMI 1640 containing 10% inactivated fetal bovine serum
The cells were cultured at 37 ° C. in a medium under the condition of 5% carbon dioxide except for the heat shock treatment.

(2) Treatment with ginsenoside Rg ₁ and heat shock In the medium of the human lung cancer cell line H69 2 days after seeding, the ginsenoside Rg ₁ (Matsuura) represented by the above formula (I) was adjusted to a final concentration of 100 μM. (Pharmaceutical industry) was added and the cells were cultured for 24 hours. Thereafter, the cells were subjected to a heat shock treatment at 45 ° C. for 15 minutes, and then cultured at 37 ° C. overnight. Control test, except that no addition of ginsenoside Rg ₁ was carried out as described above.

(3) Measurement of HSP expression level in cultured human cancer cells Each cell treated in the above section (2) was homogenized by the following method, and the HSP expression level was measured by Western blotting. That is, the cells treated in the above (2) were treated with phosphate buffered saline [composition: KCl = 0.2 g /
1, KH ₂ PO ₄ = 0.2 g / l, NaCl = 8 g /
1, Na ₂ HPO ₄ (anhydrous) = 1.15 g / l;
PBS (-)), and then lysed buffer (lysis buffer) [1.0% NP-4
0, 0.15 M sodium chloride, 50 mM Tris-HC
1 (pH 8.0), 1 mM of 5 mM-EDTA, 2 mM-N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride, 2 μg / ml leupeptin and 2 μg / ml pepstatin], and allowed to stand on ice for 20 minutes.
Thereafter, centrifugation was performed at 12,000 rpm at 4 ° C. for 20 minutes. 10 μl of the supernatant after centrifugation was added to PBS (−) 790
μl, add protein assay stain (Dye Reag
ent Concentrate: Bio-Rad, Catalog No. 500-00
06) 200 μl was added. After standing at room temperature for 5 minutes, the protein was quantified by measuring the absorbance at 595 nm.

Using the sample for which protein was quantified, L
aemmli buffer system (Laemmli, NK, "Natur
e ", 283 : pp. 249-256, 1970), lysates containing equal amounts of protein were subjected to SDS polyacrylamide gel electrophoresis. After electrophoresis, blotting and subsequent blocking were performed. Using a transfer device (Trans-Blot Electrophoretic Transfer Cell: Bio-Rad, Catalog No. 170-3946), adhere the gel to a 0.45 μm nitrocellulose membrane (Schleicher & Schuell, Catalog No. 401196) at room temperature and 100 V at room temperature. A tris glycine buffer (Tris Gly Running and Bloom) containing 0.025 M Tris and 0.192 M glycine and adjusted to pH 8.5 was used as a blotting buffer.
tting Buffer; Enprotech, Massachusetts, USA
A buffer prepared by adding methyl alcohol to catalog number SA100034) to 20% was used. After blotting, the nitrocellulose membrane was added to a 10% skim milk (Snow Brand Milk Products) -PBS (-) solution at room temperature for 30 minutes.
Incubate to block non-specific binding.

After blocking, anti-human HSP60 mouse monoclonal antibody (Stre
ssGen, Victoria, BC, Canada, Cat.No. SPA-8
06), a primary antibody reaction was performed. This anti-human HSP
60 murine monoclonal antibody is an antibody to human HSP60 prepared was prepared as an immunogen by recombinant DNA methods using E. coli ( "J. Exp. Med." 175
, 1805-1810, 1992), mammalian HSP60 (primate HS)
P60, mouse HSP60, rat HSP60, and hamster HSP60) ("J. Exp. M
ed. " 175 , 1805-1810, 1992). This anti-human HSP60
The epitope recognized by the mouse monoclonal antibody is located in the amino acid sequence consisting of amino acid residues 383 to 447 of the human HSP60 amino acid sequence ("J. Exp. Med." 175 , 1805-1810, 1992). ). After the primary antibody reaction, the solution was exchanged with PBS (-) for 5 minutes each, and the washing was performed twice with a slow rocking shaker, and PBS (-)-0.1% Tween 20 (Bio-Rad, Catalog No.). 170-6531) The solution was replaced for 15 minutes, and the solution was washed four times. Finally, the cells were washed twice with PBS (-) for 5 minutes each.

After the washing was completed, a peroxidase-labeled goat anti-mouse IgG antibody (CAPPEL, Catalog No. 55550) was
% For 2 hours using 5 ml of an antibody solution prepared by diluting 5000-fold with a PBS (-) solution containing 5% skim milk.
The next antibody reaction was performed. After the completion of the reaction, the nitrocellulose membrane was washed twice with a PBS (-) solution for 5 minutes while changing the solution twice, and further with a PBS (-)-0.1% Tween20 solution for 15 minutes for 5 times. Performed by slow rocking shaker. Finally PBS (-)
The solution was washed twice for 5 minutes each. Extra PBS
(-) After removing the solution, the western blotting detection reagent (ACL Western blotting detection reagent; Ame
rsham, catalog number RPN2106), sprinkle onto the nitrocellulose membrane, incubate for 1 minute, remove excess detection reagent, wrap the nitrocellulose membrane in wrap, and cover the reaction surface with X-ray film (Kodak X-OMAT, AR, (Catalog No. 165 1454), exposed, developed and HSP6
The existence of 0 was examined.

Control test, ie ginsenoside Rg
_The lung cancer cell line H69 without addition of ₁ had a molecular weight of about 6
One band of 0 kD was detected. The molecular weight was determined by binding to the anti-human HSP60 mouse monoclonal antibody and molecular weight markers (egg white ovalbumin and bovine serum albumin). Ginsenoside Rg ₁
In the lung cancer cell line H69 to which was added, the corresponding band was an extremely thin band. That is, ginsenoside R
It can be concluded that g ₁ has the activity of a synthetic inhibitor that suppresses the expression of HSP60.

[0032]

INDUSTRIAL APPLICABILITY As described in detail above, ginsenosides, especially ginsenoside Rg _1, are effective for intracellular HSP60.
It has the activity of a synthetic inhibitor that suppresses the expression of proteins belonging to the family. Therefore, ginsenosides, especially by administering ginsenoside Rg _1, for example,
It is possible to effectively improve the physiological condition of a patient with an autoimmune disease (for example, type I diabetes, rheumatoid arthritis, etc.) in which a protein belonging to the HSP60 family is involved in the onset, and to effectively treat the disease.

Claims (4)

[Claims] 1. A molecular weight of 57 kilodaltons to 6 containing ginsenosides as an active ingredient.
Inhibitor of heat shock protein synthesis up to 8 kilodaltons. 2. Ginsenosides are ginsenoside Rg.
The heat-shock protein synthesis inhibitor having a molecular weight of 57 to 68 kilodaltons according to claim _1, which is ₁ . 3. A heat shock protein synthesis inhibitor having a molecular weight of from 57 kilodaltons to 68 kilodaltons, which comprises as an active ingredient a plant extract containing ginsenosides. 4. A heat-shock protein synthesis inhibitor having a molecular weight of 57 to 68 kilodaltons, which contains an extract of carrot or ginseng as an active ingredient.