JPH1045572A - Magnolol-containing synthetic suppressor of protein belonging to hsp27 family - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject medicine capable of specifically suppressing synthesis of a specific heat shock protein in a cell of a tissue exhibiting a morbid state and treating cancers, multiple sclerosis, etc., by including magnolol which is an ingredient of Magnoliae Cortex as an active ingredient. SOLUTION: This medicine contains magnolol as an active ingredient. The magnolol is included in a crude medicine such as Magnoliae Cortex, which is a bark of Magnolia obovata Thunberg or Magnolia biloba (Rehd. et Wils.) Cheng. Since a material containing magnolol as an active ingredient is effective, crude extract of Magnoliae Cortex can be used. The extract of Magnoliae Cortex is obtained by extracting Magnoliae Cortex with water (e.g. cold water, warm water or hot water) or an organic solvent. Thereby, synthesis of heat shock protein having 16-40K dalton molecular weight in the cell can specifically be suppressed, the medicine can be used for treating and preventing a morbid state on which the protein is adversely influenced.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a process for producing magnolol as an active ingredient, which has a molecular weight of 16 kilodaltons (k).
The present invention relates to a synthesis inhibitor of a protein belonging to the group of heat shock proteins between D) and 40 kD (hereinafter, referred to as HSP27 family). HSP27 according to the invention
Synthetic inhibitors of proteins belonging to the family, in particular,
By suppressing the tissue synthesis of the proteins belonging to the HSP27 family, the proteins belonging to the HSP27 family are thought to be involved in the onset, malignant transformation, or impaired treatment, such as cancer or multiple sclerosis. Etc. can be effectively improved to effectively treat the above-mentioned diseases.

[0002]

BACKGROUND OF THE INVENTION Despite recent advances in chemotherapy, surgery, radiation therapy, immunotherapy, etc., the cause of death due to cancer is still directly or indirectly related to the malignant transformation of cancer. Overcoming malignancy has become one of the major challenges in cancer treatment in the future. The malignancy of a cancer is determined by the proliferative, invasive, or metastatic nature of the cancer. Metastasis, which is one of the phenomena of malignant transformation, depends on the type of primary cancer, and the organs that are prone to metastasis vary. Cancer metastasis is a complex event that begins with the growth of the primary tumor, withdrawal of cancer cells from the primary focus and invasion / proliferation of surrounding tissues, tumor angiogenesis, invasion of cancer cells into nearby blood vessels, blood New tumor blood vessels are born, which is visible after the movement to the distant site by flow and adhesion / implantation to vascular endothelial cells, infiltration outside the blood vessel, and the initiation of proliferation at the distant site (metastasis). It consists of a complex reaction cascade leading to the formation of specific metastatic foci. Generally, cancer is divided into those with high malignancy and those with relatively low malignancy. However, no radical treatment has been established for highly aggressive cancers, and patients are most likely to eventually die.

[0003] Hyperthermia for cancer (hyperthermia;
Hyperthermia) is a method of selectively killing tumor cells by heating a cancer tissue to treat cancer, and has recently attracted attention. Cancer treatment with hyperthermia
Looking at the biological effects of heat, a cell-killing effect can be obtained with relatively mild heating of 41 to 45 ° C, and a synergistic effect can be obtained when used in combination with radiation or an anticancer agent. There are many advantages. Treatment of cancer by hyperthermia has been tried in almost all departments in the clinic. However, one of the problems of hyperthermia is heat resistance which is transiently induced after heating. That is, since the cancer cells temporarily become thermotolerant by the first heating, the cell killing effect by the next heating is reduced. The thermotolerance means that once a cell (or tissue) is heated sub-lethal once, the cell (or tissue) becomes transiently heat resistant to the next heating. Due to heat resistance, the current practice is that thermal treatment is performed in most facilities only once or twice a week.

[0004] In cancer chemotherapy, there are still solid cancers such as lung cancer and colon cancer which hardly respond to chemotherapy. Is a problem. 1
According to United States statistics of 988, 49% of cancers diagnosed in one year are intrinsically resistant to chemotherapy initially, and 47% were initially chemotherapy effective and the tumor regressed It is said that the acquired resistance has recurred.
These facts indicate that one of the most important problems that hinders the effects of chemotherapy on cancer is resistance to cytotoxic drugs.

In addition, multiple sclerosi
s, MS) is an inflammatory demyelinating disease that specifically impairs the central nervous white matter, and its pathogenesis has been demonstrated by the immune system to attack the myelin sheath that surrounds nerve fibers. It is an immune disease. Multiple sclerosis often repeats acute hatred / remission in the early stages, but then gradually progresses. Adrenocorticotropic hormone (ACTH) and corticosteroids are used to improve symptoms during the acute phase, and azathioprine and cyclophosphamide are used to prevent recurrence during remission and to prevent chronic progression of symptoms. Immunosuppressive drugs such as sac have been used. However, many of the drugs currently being administered to multiple sclerosis patients are not as effective as expected,
The current situation is that it is not enough, as non-specific therapy has many side effects. Development of a more specific treatment method for multiple sclerosis is expected.

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

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 number described immediately after the HSP. For example, an HSP having a molecular weight of 27 kD is referred to as “HSP27”. As described above, there are many kinds of HSPs, but they differ not only in molecular weight but also in structure, function, or property. In addition to stress response, some of these proteins are constitutively synthesized and, under normal circumstances, are involved in protein folding, unfolding, protein subunit association, and protein membrane transport. It has been shown to play an essential physiological role. These functions as heat shock proteins are called molecular chaperones.

Expression of proteins belonging to the HSP27 family is significantly correlated with lymph node metastasis, lymphatic and vascular invasion, and shorter survival rate in human breast cancer ("J. Natl. Cancer Inst."). 83 : 170-178, 1991). It has been reported that a protein belonging to the HSP27 family is also a negative prognostic factor in gastric cancer ("Br.J.
Surg. ", 78 : 334-336, 1991). There is also a report that the expression level of proteins belonging to the HSP27 family in primary cancer cells is positively correlated with cancer malignancy, especially the recurrence rate of cancer (" Breast Cancer "). Res. Treat. ", 12 : 130,
1988; "Proc. Am. Assoc. Cancer Res.", 30 : 252, 19
89) By suppressing the expression of proteins belonging to the HSP27 family, malignant transformation of cancer can be prevented.

HS for heat resistance, which is a problem in hyperthermia of cancer
There are reports that proteins belonging to the P27 family are involved. When the human HSP27 gene was introduced into mouse or hamster cells and expressed, the number of heat-resistant cells surviving after heat shock was induced and increased depending on the amount of HSP27 protein ("J. Cell. Biol.", 109
: 7-15, 1989). In addition, in Chinese hamster cells, a mutant strain that constantly expresses HSP27 becomes able to acquire heat resistance ("J. Cell. Physio").
l. ", 137 : 157, 1988) α-B crystallin is a protein which is induced by heat shock treatment and has a high homology in amino acid sequence with HSP27, and is one of the proteins belonging to the HSP27 family. Cells overexpressing -B crystallin also acquire resistance to heat stress ("J. Cell. Biol.", 125 : 1385-1393, 1994), which suppresses the expression of proteins belonging to the HSP27 family. This has shown the possibility of suppressing hyperthermia and enhancing the effect of hyperthermia on cancer, and has also been reported to correlate the expression of proteins belonging to the HSP27 family with drug resistance ("Breast Ca
ncer Res. Treat. ", 23 : 178, 1992;" Cancer Res. ", 5
1 : 5245-5252, 1991), it is also possible to suppress drug resistance and enhance the effect of chemotherapy by suppressing the expression of proteins belonging to the HSP27 family.

The immunodominant antigen in multiple sclerosis is αα, a protein belonging to the HSP27 family.
-B crystallin ("N
Nature ", 375 : 739-740, 1995). The expression of α-B crystallin in nervous tissue of multiple sclerosis patients is stronger than that in non-diseased tissues and is very immunogenic. Is high ("Nature", 375 : 798-801, 1995). These facts
HSP27 is the autoantigen in multiple sclerosis
This is α-B crystallin, which is one of the proteins belonging to the family, and it has been shown that suppressing the expression of α-B crystallin in the myelin sheath is linked to the fundamental treatment of multiple sclerosis.

[0011]

SUMMARY OF THE INVENTION In view of the above circumstances, the present inventors effectively improve the physiological condition of a patient suffering from a disease such as cancer or multiple sclerosis, and effectively treat the disease. In order to develop a possible method, various studies have been conducted on compounds showing a synthetic inhibitory action on proteins belonging to the HSP27 family. As a result, the present inventors have surprisingly found that magnolol, which is a component of pearl oyster, specifically suppresses the synthesis of proteins belonging to the HSP27 family in cells of tissues showing pathological conditions. That is, it has been found that administration of magnolol suppresses the synthesis of a protein belonging to the HSP27 family in cells, and thus can treat diseases such as cancer and multiple sclerosis. The present invention is based on such findings, and an object of the present invention is to provide a synthesis inhibitor of a protein belonging to the HSP27 family, which can effectively treat diseases such as cancer and multiple sclerosis.

[0012]

Accordingly, the present invention is characterized by containing magnolol as an active ingredient,
It relates to inhibitors of the synthesis of heat shock proteins with a molecular weight between 16 and 40 kilodaltons.

As used herein, the "HSP27 family" has a molecular weight of 16 kD to 40 kD.
Of heat shock proteins. Examples of proteins belonging to the HSP27 family include mammalian HSP27 (ie, a heat shock protein having a molecular weight of 27 kD) [or HSP28 (ie, a heat shock protein having a molecular weight of 28 kD)] and avian HSP2
5 (ie, a heat shock protein with a molecular weight of 25 kD) or yeast HSP26 (ie, a molecular weight of 26 kD).
D heat shock protein). Generally, the molecular weight of a protein is, for example,
Since some differences occur due to differences in molecular weight measurement methods, experimental conditions, and the like, some proteins belonging to the HSP27 family include HSP27 and HSP27 in mammals.
Even though the molecular weights are different, such as SP28, it is not clear at present whether they are different proteins having different amino acid sequences, or whether they are the same proteins that differ only in the molecular weight. Some are not included. The protein belonging to the HSP27 family is the most major heat shock protein in mammals among the heat shock proteins belonging to the small HSP family described above, and exhibits characteristics that are well conserved across species. However, proteins belonging to the HSP27 family are different from other heat shock proteins and have different molecular weights for each species, and have a molecular weight of 16 k
It is a very diverse protein with D to 40 kD. Α-B crystallin having a high amino acid sequence homology with HSP27 is also induced by heat shock treatment and is one of the proteins belonging to the HSP27 family.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The synthesis inhibitor of the present invention contains magnolol as an active ingredient. Magnolol which can be used as an active ingredient in the synthetic inhibitor of the present invention has the formula (I):

Embedded image The compound is represented by the formula (1), and is contained in crude drugs such as red pearls.

The magnolol 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 magnolol used as an active ingredient in the synthesis inhibitor of the present invention is extracted from a natural product, for example, whole or a part of a plant containing magnolol (for example, whole plant, leaf, root, rhizome, stem,
Root bark, flower, or fruit) can be used as is or simply processed (eg, 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 the plant containing magnolol include, but are not limited to, Magnolia obovata Thumb.
erg), Magnolia biroba [Magnolia b
iloba (Rehd. et Wils.) Chen
g], Calla hoo (Magnolia Magnolia o)
ficinalis Rehd. et Wil
s. ) Or Magnolia splengeri (Magnol)
ia sprengeriPamp. ) Etc. can be used.

When magnolol in the present invention is extracted from a crude drug, it is not limited to this, but for example,
It is preferable to extract from pearl oysters. Koboku (Makol; Magnoliae Cortex; Magnol)
ia Bark is the bark of Japanese cypress (Wakoboku;
Japanese bark) or bark of Karahoo (Karakoboku; Karako)
And these parts can be used alone or in any combination.

The Komoku extract that can be used as an active ingredient in the synthesis inhibitor of the present invention only needs to contain the above-mentioned magnolol, and therefore, a rough extract of Komoku can be used. As a method for producing the Konokoku extract that can be used in the present invention, Konokoku can be obtained by extracting Konokoku with water (for example, cold water, hot water, or hot water) or an organic solvent. . As the organic solvent, for example, carbon number 1 to
6, alcohols (eg, methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol, or butyl alcohol), esters (eg, methyl acetate, ethyl acetate, propyl acetate, or butyl acetate), ketones (eg, acetone or methyl) Isobutyl ketone), ether, petroleum ether, n-
Hexane, cyclohexane, toluene, benzene, a halogen derivative of a hydrocarbon (for example, carbon tetrachloride, dichloromethane, or chloroform), pyridine, glycol (for example, propylene glycol or butylene glycol), polyethylene glycol, acetonitrile, or the like can be used. It is possible to use these organic solvents alone or in appropriate combination, mix them at a fixed ratio, and further use them in an anhydrous or water-containing state. As a method of water extraction or organic solvent extraction, a method used for ordinary crude drug extraction can be used. For example, with respect to (dry) 1 part by weight of kouboku, using 3 to 300 parts by weight of water or an organic solvent, While stirring, heat reflux at a temperature below its boiling point,
Ultrasonic extraction or cold immersion at room temperature is desirable.
The extraction step is usually performed for 5 minutes to 7 days, preferably for 10 minutes to
The extraction is performed for 60 hours, and the extraction time can be reduced by adding auxiliary means such as stirring as necessary.

After completion of the extraction step, the insoluble matter can be separated from the water or organic solvent extract by a suitable method such as filtration or centrifugation to obtain a crude extract. In the synthesis inhibitor of the present invention, when magnolol extracted and fractionated from a natural product is used, the crude extract may be used as it is without further purification. In addition to the water extract or organic solvent extract by a conventional method, a purified extract obtained by further treating the above crude extract with various organic solvents or adsorbents is also used as an active ingredient of the synthesis inhibitor of the present invention. You can As for the red pearl extract containing these crude extracts and purified extracts that have undergone various purification treatments, the solution as extracted may be used, or an extract obtained by concentrating the solvent may be used, or the solvent is distilled off. A dry powder, a crystallized and purified product, or a viscous substance may be used, or a diluted solution thereof may be used. The kelp extract thus obtained contains magnolol contained in kelp, and at the same time, contains impurities derived from kelp as a raw material.

The synthesis inhibitor of the present invention is a magnolol or an extract of a plant containing magnolol, for example, an extract of a herbal medicine containing magnolol (in particular, an extract of magnolia) alone, Or preferably with a conventional carrier which is pharmaceutically or veterinarily acceptable,
It can be administered to animals, preferably mammals (especially humans). The dosage form is not particularly limited. For example, oral preparations such as powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts, or pills, or injections And parenteral agents such as topical solutions, topical solutions, ointments, suppositories, creams for topical administration, and eye drops. These oral agents are, for example,
Gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soy lecithin, sucrose, fatty acid ester, talc, magnesium stearate, polyethylene glycol, magnesium silicate. Excipients such as silica, anhydrous silicic acid or synthetic aluminum silicate, binders, disintegrants, surfactants, lubricants, fluidity promoters, diluents, preservatives, coloring agents, flavors, flavoring agents, stabilizing agents. It can be produced according to a conventional method using an agent, a humectant, a preservative, an antioxidant, or the like. For example, 1 part by weight of magnolol and 9 lactose
And 9 parts by weight of a capsule.

Examples of the parenteral administration method include injection (subcutaneous, intravenous, etc.), rectal administration and the like. Among these, the injection is most preferably used. For example, in the preparation of injectables, magnolol as an active ingredient, or a plant extract containing magnolol, for example, in addition to a crude drug extract containing magnolol (particularly, kokuboku extract), For example, water-soluble solvents such as physiological saline or Ringer's solution, non-water-soluble solvents such as vegetable oils or fatty acid esters, isotonic agents such as glucose or sodium chloride, solubilizing agents, stabilizers, preservatives, suspending agents. , Or an emulsifier can be optionally used. In addition, the synthetic inhibitor of the present invention may be administered by the method of sustained release preparation 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 can contain, but is not limited to, magnolol in an amount of 0.01 to 99% by weight, preferably 0.1 to 80% by weight. . Further, a plant extract containing magnolol, for example, a synthetic inhibitor of the present invention containing an extract of a herbal medicine containing magnolol (particularly, an extract of magnolia) as an active ingredient is included therein. Magnolol can be prepared by appropriately adjusting the amount of magnolol to fall within the above range. Incidentally, a plant extract containing magnolol,
For example, when a synthetic inhibitor containing an extract of a crude drug containing magnolol (especially an extract of Asparagus) as an active ingredient is used as a formulation for oral administration, a pharmaceutically acceptable carrier can be used. It is preferable to formulate the compound using The dose when the synthetic inhibitor of the present invention is used is
There is no particular limitation, depending on the type of illness, age, sex, weight of the patient, degree of symptoms, administration method, etc., but there is no particular limitation, but the amount of magnolol is usually 1 mg to 10 g per adult 1 to 4 times a day. It is orally or parenterally administered to some extent. Furthermore, the application is not limited to pharmaceuticals, and various applications such as functional foods and health foods can be given in the form of food and drink.

[0022]

As described above, the magnolol contained in the synthetic inhibitor of the present invention has an action of specifically suppressing the synthesis of intracellular proteins belonging to the HSP27 family. HSP2 at
Biosynthesis of proteins belonging to 7 families is specifically reduced. Therefore, the magnolol is, for example, HSP
Proteins belonging to the 27 family prevent and treat cancer associated with its malignant transformation, proteins belonging to the HSP27 family enhance the effect of cancer hyperthermia associated with hyperthermia, which is an obstacle to the therapy, or proteins belonging to the HSP27 family Can be used for the prevention and treatment of autoimmune diseases such as multiple sclerosis associated with its onset. In addition, there is a report that the expression of proteins belonging to the HSP27 family is correlated with drug resistance ("Breast Cancer Res. Treat.", 23 : 178, 1992; "Ca
ncer Res. ", 51 : 5245-5252, 1991), by suppressing the expression of proteins belonging to the HSP27 family,
It is also possible to suppress drug resistance and enhance the effect of chemotherapy.

[0023]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. Example 1: Measurement of HSP expression level of human cultured cancer cells (1) Culture of human cultured cancer cells The following human cultured cancer cells were cultured at 37 ° C under 5% carbon dioxide conditions except for heat shock treatment. Cultured. Lung cancer cell line H69 (ATCC HTB 119) is 10%
RP containing inactivated fetal bovine serum (hereinafter referred to as FBS)
Cultured in MI1640 medium. Uterine cancer cell line HeLa
S3 (ATCC CCL 2.2) was cultured in MEM medium containing 10% inactivated FBS.

(2) Magnolol treatment and heat shock treatment Two days after seeding, in the culture medium of the lung cancer cell line H69, the magnolol represented by the above formula (I) was adjusted to a final concentration of 100 μM (Matsuura Pharmaceutical) Was added and the cells were cultured for 24 hours. Magnolol (Matsuura Pharmaceutical Co., Ltd.) represented by the above formula (I) was added to the medium of the uterine cancer cell line HeLa S3 two days after the seeding so as to have a final concentration of 20 μM, and the cells were cultured for 24 hours. Then, after heat shock treatment at 45 ° C. for 15 minutes, the cells were cultured at 37 ° C. overnight. The control test was performed as described above except that no magnolol was added.

(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, 0.15M sodium chloride, 50 mM Tris-HC
1 (pH 8.0), 5 mM-EDTA, 2 mM-N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride, 2 μg / ml leupeptin and 2 μg / ml pepstatin] were added, and the mixture was 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
In addition to μl, 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 absorbance was measured at 595 nm for protein quantification.

Using the sample for which the protein was quantified, L
aemmli buffer system (Laemmli, NK, "Natur
e ", 283 : pp. 249-256, 1970), SDS polyacrylamide gel electrophoresis of a lysate containing an equal amount of protein was performed. 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 100 V at room temperature. Blotting was carried out for 3 hours, and the blotting buffer was Tris Gly Running and Blos (0.025 M Tris and 0.192 M glycine) adjusted to pH 8.5.
tting Buffer; Enprotech, Massachusetts, USA,
A buffer prepared by adding 20% methyl alcohol to catalog number SA100034) 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, an anti-human HSP27 mouse monoclonal antibody (Stre
ssGen, Victoria, BC, Canada, Catalog Number SPA-8
00), a primary antibody reaction was performed. This anti-human HSP
The 27 mouse monoclonal antibody is a human breast cancer cell line MC
HSP2 isolated from F7 (ATCC HTB 22)
4 was an immunogen produced by using "Cancer Res.",
42 , 4256-4258, 1982), HSP27 (human HSP2)
7, chimpanzee HSP27, and sheep HSP27)
("Cancer Res.", 42 , 4256-4258, 1
982; "Cancer Res.", 43 , 4297-4301, 1983), HSP
It also reacts specifically with 24 and HSP28. After the primary antibody reaction, the solution was exchanged with PBS (-) for 5 minutes each for 2 minutes.
Washing is performed by a slow rocking shaker, and further, washing is performed four times by replacing the solution with a PBS (−)-0.1% Tween 20 (Bio-Rad, Cat. No. 170-6531) solution for 15 minutes. Was. Finally,
Washing was performed twice for 5 minutes each with PBS (-).

After the washing was completed, a peroxidase-labeled goat anti-mouse IgG antibody (CAPPEL, Catalog No. 55550) was
Using 5 ml of antibody solution prepared by diluting 5000 times with PBS (-) solution containing% skim milk, 2 hours, 2
A secondary antibody reaction was performed. After completion of the reaction, the nitrocellulose membrane was washed twice with a PBS (-) solution for 5 minutes each, and for 5 times with a PBS (-)-0.1% Tween 20 solution for 15 minutes each. 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, Cathode No. 165 1454) exposed to light, developed and HSP2
7 was examined. Table 1 shows the results. In the table,
"↓" indicates that HS is higher due to magnolol treatment than control.
It means that the P27 expression level was decreased.

[0029]

[Table 1] Cancer type Cancer cell Uterus HeLa S3 ↓ Lung H69 ↓

Control tests, ie uterine cancer cell line HeLaS3 and lung cancer cell line H without addition of magnolol
In 69, one band having a molecular weight of about 27 kD was detected. The molecular weight was determined by binding to the anti-human HSP27 mouse monoclonal antibody and molecular weight markers (soybean trypsin inhibitor and bovine carbonic anhydrase). In the uterine cancer cell line HeLa S3 supplemented with magnolol, a band with a molecular weight of about 27 kD was not detected. In the lung cancer cell line H69 to which magnolol was added, the concentration of the band having a molecular weight of about 27 kD was significantly lower than that in the control test. That is, it can be concluded that magnolol has the activity of a synthetic inhibitor that suppresses the expression of HSP27.

[0031]

As described in detail above, magnolol is
It has the activity of a synthetic inhibitor that suppresses the expression of proteins belonging to the HSP27 family in cells. Therefore, by administering magnolol, for example, a protein belonging to the HSP27 family may be associated with malignancy or a decrease in the effects of hyperthermia, or a cancer associated with the onset thereof, or a patient with an autoimmune disease such as multiple sclerosis associated with the onset thereof. The physiological condition can be effectively improved, and the disease can be effectively treated.
In addition, there is a report that the expression of a protein belonging to the HSP27 family is correlated with drug resistance ("Breast Ca
ncer Res. Treat. ", 23 : 178, 1992;" Cancer Res. ", 5
1 : 5245-5252, 1991), by suppressing the expression of proteins belonging to the HSP27 family, it is also possible to suppress drug resistance and enhance the effect of chemotherapy.

Claims (3)

[Claims] 1. A heat shock protein synthesis inhibitor having a molecular weight of 16 to 40 kDa, comprising magnolol as an active ingredient. 2. A composition comprising a plant extract containing magnolol as an active ingredient and having a molecular weight of 16
Inhibitor of heat shock protein synthesis between kilodalton and 40 kilodalton. 3. A composition having a molecular weight of from 16 kilodaltons to 4 kilograms, characterized by containing an extract of Aspergillus niger as an active ingredient.
Inhibitor of heat shock protein synthesis up to 0 kilodaltons.