KR100591164B1 - Herbal extract for prophylaxis or treatment of inflammatory diseases and process for preparation thereof - Google Patents

Abstract

The present invention relates to herbal extracts for the prophylaxis or treatment of inflammatory diseases and methods for their preparation. Specifically, the present invention provides a water or alcohol extract of a mixture of pine, gingiber, dew, organo and licorice mixtures for the prevention or treatment of inflammatory neurological diseases such as degenerative arthritis, rheumatoid arthritis, arthritis due to physical damage and It relates to a composition.

Description

Herbal extract for the prevention or treatment of inflammatory diseases and preparation method thereof {HERBAL EXTRACT FOR PROPHYLAXIS OR TREATMENT OF INFLAMMATORY DISEASES AND PROCESS FOR PREPARATION THEREOF}

The present invention relates to herbal extracts for the prophylaxis or treatment of inflammatory diseases, and methods of preparing the same, and more particularly, to inflammatory diseases, including water extracts or alcohol extracts of a mixture of cypress, oleander, dew, cucumber and licorice as an active ingredient, in particular The present invention relates to a composition for preventing or treating inflammatory neurological disease, for example, degenerative arthritis, rheumatoid arthritis, arthritis or inflammatory pain due to physical damage, and a method of manufacturing the same.

Joints consist of articular cartilage and the surrounding bone, ligaments, and synovial envelope that surrounds the joint. Inflammatory diseases in the joints include chronic rheumatoid arthritis, which is thought to be caused by subjective immunity, infectious arthritis caused by bacterial infections, and degenerative arthritis and connective tissue in which degeneration or destruction of articular cartilage or bone is caused by various causes. Degenerative changes can be categorized into crystalline arthritis, in which soluble metabolites are deposited as crystals in connective tissue around joints.

Degenerative changes that occur with age begin in the articular cartilage. When the chondrocytes that produce the components that make up the cartilage are aged, their function decreases, and the elasticity of the membrane and cartilage surrounding the joint decreases and the ability to protect the joint from external shocks is weakened. If this condition persists, inflammation is repeatedly caused by various substances entering the joint cavity surrounded by the surface of the cartilage and become rough. On the other hand, articular cartilage repeats the process of destruction and production, where more cartilage is destroyed than it is produced, and the amount of articular cartilage that absorbs shock is gradually reduced and even disappears. This causes the bones between the joints and the bones to come in contact with it, which causes extreme pain.

Degenerative arthritis, or osteoarthritis, is generally an inevitable disease caused by aging, and the most common joint disease and its cause are not yet clearly identified, and the general causes of arthritis are considered to be combined, and some specific causes are also considered. It is becoming. In general, the incidence of degenerative arthritis in older adults is the result of prolonged exposure to pathophysiological processes in childhood. In particular, the causes of degenerative arthritis are:

1) Aging-Aging is not the only cause of osteoarthritis, but with age, the blood flow to the joints decreases, and the rate of bone remodeling at the bone-cartilage junctions decreases. In addition, as the age of peripheral nerve function decreases, which is also considered to increase the predisposition of osteoarthritis.

2) Primary changes of cartilage stroma, cartilage cell metabolic function and metabolic regulators are also important causes of degenerative arthritis. On the other hand, recovery after mechanical damage may not be complete or may be caused by the sequelae of inflammatory joint disease.

Obesity-Obesity significantly increases the load on the weight-bearing joints, alters posture and gait, and continues to degenerative arthritis if this condition persists.

However, with the development of molecular biology, inflammatory neuro-articular diseases have been attempted at the immunological level of cytokines, and factors affecting these diseases have been identified one by one. One of them is the study of cytokines secreted by mast cells.

Mast cells are well known as cells mainly involved in allergic reactions or various inflammatory reactions. These cells secrete IL-8, IL-13, TNF-α, granulocyte macrophage colony stimulating factor (GM-CSF), IFN-x, and leukemia inhibitory factor or increase the expression of each mRNA. do. These cytokines not only regulate IgE antibody production, immune responses, but also play an important role in the development of inflammatory responses through hematopoietic cells, tissue responses, and vascular responses. Among the various cytokines secreted from mast cells, IL-1, IL-6, and TNF-α are particularly important cytokines in inducing an inflammatory response. In particular, TNF-α and IL-1 are recognized as the most important cytokines that cause rheumatoid arthritis. This implies that the regulation of these cytokines is a critical method for the treatment of rheumatoid arthritis. These cytokines stimulate the acute reactive protein genes to produce proteins and stimulate constitutive factors to activate inflammatory cells. Among them, TNF-α induces nerve loss through tissue damage and acts as a major factor in the inflammatory response. IL-6 appears to be involved in the pathogenesis of various diseases when overproduced in vivo, and is involved in the development of various malignancies, proliferative diseases, autoimmune diseases and infectious diseases. The expression of TNF-α and IL-6 among them depends on the activity of the transcription factor, NF-κB. At least five other genes, NFκB1 (p105 / 50), NFκB2 (p100 / 52), ReA (p65), Rel B and c-Rel, belong to the NF-κB family. In general, NF-κB consists of a dimer, which consists of NFκB1 (p50) and Rel A (p65) or NFκB1 (p50) and NFκB2 (p52). NF-κB binds to the endogenous inhibitor IκB and is present in the cytoplasm. When phosphorylation and degradation of IκB occurs by a specific signal, NF-κB migrates into the nucleus and specific DNA sequences present in promoters of genes such as TNF-α and IL-6 Activated by binding.

On the other hand, drugs used for the treatment of neuroarthritis can be roughly divided based on three main mechanisms of action: i) reduction of inflammation, ii) delay in disease progression and iii) reduction in uric acid concentration. Many drugs for the treatment of neuroarthritis are used to reduce inflammation. Inflammation is a pathological process that causes pain, edema, heat, redness and stiffness. Drugs that relieve inflammation rapidly include nonsteroidal anti-inflammatory drugs including aspirin and steroids such as cortisone. Aspirin, analgesics, or nonsteroidal anti-inflammatory drugs can reduce pain and ease the movement of nerve joints. However, when taking these drugs, gastrointestinal disorders or stomach pain may be caused, and the basic side effects, as well as a long time, seriously harmful effects on the human body. Therefore, these drugs are basically contraindicated in people with active peptic ulcers or hemorrhagic history of the gastrointestinal tract. In addition, the steroid hormone preparations are not used well for degenerative arthritis due to serious side effects such as weight gain and hypertension. However, intra-articular injections of steroid preparations are sometimes used when the inflammation of the knee joint is very severe and the therapeutic effects of other drugs do not meet expectations. However, steroid injections are completely irrelevant to the treatment of the cause of the disease, and can be excessively used simply by temporarily reducing pain, which can destroy nerve joints and exacerbate disorders. Therefore, there is an urgent need for the development of a new concept of drug that can effectively prevent or treat inflammatory neurological diseases.

Accordingly, the present inventors have conducted continuous research to develop a material for preventing or treating inflammatory neuron disease with no side effects, and as a result, Catalpa ovata , Caragana sinica , Achyranthes bidentata , and Acanthopanax sessiliflorus SEEM ) and licorice ( Glycyrrhiza uralensis FISCH ) mixture of water extracts or alcohol extracts can solve the problems of the prior art as mentioned above, and the present invention was completed by confirming that excellent medicinal properties can be obtained.

Summary of the Invention

It is an object of the present invention to produce novel herbal extracts and their preparations that exhibit excellent efficacy against inflammatory neurological diseases, such as degenerative arthritis, rheumatoid arthritis, neuroarthritis or inflammatory low back pain due to physical injury, without causing side effects, and the preparation thereof. To provide a way.

1 is a graph showing the effect of the subject composition of the invention on the viability of cells in HMC-1 cells,

A of FIG. 2 is a graph showing the secretion of TNF-α with time in the HMC-1 cells,

B of FIG. 2 is a graph showing the effect of the subject composition of the present invention on the secretion of TNF-α in the HMC-1 cells,

A of FIG. 3 is a graph showing the secretion of IL-1β according to the time in the HMC-1 cells,

B of FIG. 3 is a graph showing the effect of the subject composition of the present invention on the secretion of IL-1β in the HMC-1 cells,

A of Fig. 4 is a graph showing the secretion of IL-8 in accordance with the time in the HMC-1 cells,

B of FIG. 4 is a graph showing the effect of the subject composition of the present invention on the secretion of IL-8 in the HMC-1 cells,

5 is an electrophoretic photograph showing the effect of the subject composition of the invention on the expression of TNF-α, IL-1β and IL-8 in HMC-1 cells.

A: Expression of TNF-α mRNA after 7 hours after PMA stimulation

B: Expression of IL-1β mRNA 5 hours after A23187 stimulation

C: Expression of IL-8 mRNA 5 hours after A23187 stimulation

In order to achieve the above object, the present invention provides a water extract or an alcohol extract of a mixture of gingko, oleander, dew, cucumber and licorice for the prevention or treatment of inflammatory diseases.

The present invention also provides a subject composition for the prophylaxis or treatment of inflammatory diseases obtained by fermentation after adding a mixture of rice, koji or yeast or strain isolated from yeast to the extract.

The present invention also provides a method of preparing the extract and the subject composition.

The present invention also provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases containing the extract or the subject composition as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides water extracts or alcohol extracts of a mixture of gingko, oleander, dew, cucumber and licorice for the prevention or treatment of inflammatory diseases.

In the above, the alcohol may be selected from the group consisting of 10 to 100% of methanol, ethanol, propanol and butanol, and it is preferable to extract for 5 to 10 hours by adding 70-99.9% of ethanol.

In the extract of the present invention, it is preferable that the raw materials of the extract include hawthorn, oleander, hyssop, scabbard and licorice in a weight ratio of 0.5 to 1: 0.5 to 1: 0.5 to 1: 0.1 to 1: 0.05 to 0.1.

The composition ratio of the above-mentioned herbal medicines is obtained based on the repeated test results, and when the lower limit is lower than the lower limit, the pharmacological effect of the component may be reduced, and when higher than the upper limit, the pharmacological effect of the other components may be reduced. There is a fear that cooperation will be reduced.

The young tree, which is the first active ingredient of the present invention, belongs to the family Jacarinaceae and is also known as a paulownia tree. It is a large leafy tree, about 6 m in height, leaves are broad grain, bark is grayish white, and in July, pale yellow flowers bloom in the end of cone branch in turn. It is said that the main tree is called the king of 100 trees. It is considered to be very spiritual as a tree that does not fall thunderbolt, and it is also called as a brain tree, a brain movement, etc., and it is mainly used as a wood for palaces and temples. In the private sector, it was traditionally used for nephritis, peritonitis, uremia, edema, and as a diuretic ingredient. In recent years, it has been applied in the private sector as an anticancer medicine such as liver cancer, cirrhosis, leukemia.

The second active ingredient of the present invention goldamcho belong to the legumes and the traditional Chinese medicine called kumjakeun root has been used as the root of the medicinal herbs. For example, it has a diuretic effect and can be applied to weak constitution or edema. In addition, it has been recorded that the symptoms of customs and soreness, because it has the effect of the blood and pain.

The third active ingredient of the present invention is the root of the herb is a horse knee which belongs to Viromaceae. It has been used for symptom of blood menopause, dysmenorrhea and dysmenorrhea because of its activity.

Ogapi, the fourth active ingredient of the present invention, is the bark or bark of the deciduous juniper tree belonging to the Oga family, and the roots or bark of the same root plant. There is a record of taking it.

Licorice, which is the fifth active ingredient of the present invention, is a perennial herb and roots of licorice, and functions as bovine ripening, clear heat detoxification, and lubricity. This harmonizes the various drugs to relieve the mild properties of the drugs.

The present invention also provides a subject composition obtained by adding fermentation to the water extract or alcohol extract after addition of a mixture of rice, yeast or yeast, and a strain isolated from the yeast.

In the composition of the present invention, it is preferable that the oak, gallweed, hyssop, cucumber and licorice are mixed in a weight ratio of 0.5 to 1: 0.5 to 1: 0.5 to 1: 0.1 to 1: 0.05 to 0.1, and here, rice and yeast And a main hair mixed in a weight ratio of 1 to 4: 0.2 to 1: 0.02 to 0.1. The composition of the present invention is preferably formulated in a liquid formulation.

The present invention also provides a method for preparing the water extract or alcohol extract and the subject composition.

The method for producing water extract or alcohol extract of the present invention

1) heating by adding water or alcohol to a mixture of cypress, gallweed, dew, organza and licorice; And

2) After separating the liquid part and the solid part consists of the step of concentrating the liquid part.

In the above, the alcohol may be selected from the group consisting of 10 to 100% of methanol, ethanol, propanol and butanol, and is preferably extracted for 5 to 10 hours by adding 70-99.9% of ethanol.

The method for preparing the subject composition of the present invention

1) fermenting the water extract or alcohol extract by adding a mixture of rice, yeast or strains separated from the yeast and the mother seed; And

2) After separating the liquid part and the solid part consists of the step of concentrating the liquid part.

In the above, the rice may be used in general, but it is preferable to use the rice.

The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease containing the water extract or alcohol extract or the subject composition as an active ingredient.

Inflammatory diseases in which the pharmaceutical composition of the present invention is effective are inflammatory neurological diseases, which may be selected from the group consisting of degenerative arthritis, rheumatoid arthritis, neuroarthritis due to physical injury, and inflammatory low back pain, but are not necessarily limited thereto. The composition of the present invention regulates the production of cytokines and chemical mediators from immune cells to inhibit local and systemic inflammatory responses (see Experimental Examples 1 to 3).

The pharmaceutical composition of the present invention may be prepared by mixing the herbal medicine, or extracting the herbal medicine according to the physicochemical properties of the active ingredient or the powder obtained by concentrating and drying the herbal medicine together with a pharmaceutically acceptable carrier. It can be prepared into pills, granules, liquids and the like using the pharmaceutical production method. The composition of the present invention is preferably in the form of a liquid, but if necessary, it may be prepared in the form of pills, granules, sprays, objects, tablets or capsules, and, if necessary, may be used in other dosage forms. In addition, when the composition of the present invention is prepared in a suitable concentration of the subject matter, it is most preferable to prepare a liquid formulation.

The composition of the present invention is poured into a mixture of, for example, 0.5-1 kg of cypress, 0.5-1 kg of gallweed, 0.5-1 kg of dew, 0.1-1 kg of cucumber, and 0.05-0.1 kg of licorice, for about 2.5 hours The extract is concentrated to about 8,000 ml to obtain an extract, and the extract is poured into a homogeneous mixture of steamed rice made from 1 to 4 kg of rice, 0.2 to 1 kg of yeast, and 0.02 to 0.1 kg of hair seedling, and 3 to 3 at 20 to 27 ° C. Fermentation was carried out for 10 days and the subject matter was prepared by filtration, and then adjusted to 10,000 ml by adding purified water. A typical single dose of the composition of the present invention is 1.5 to 2.5 ml per kg of body weight, and it is preferable to take it three times a day, but the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age of the patient These may be appropriately selected depending on the sex, condition and severity of the disease to be treated.

For example, for an adult weighing 60 kg, take 90-150 ml once three times a day. When taken at this dose, the preparation is about 20 to 25 days old. However, the composition of the present invention can be appropriately increased or decreased the dose depending on the weight, age, sex, severity of the patient and the digestive state. In other formulations, an appropriate amount calculated in consideration of the liquid dosage can be administered orally or parenterally. As early as 2-3 days after taking the symptoms may reduce the symptoms, but there are individual differences. Take it for about a month after the effect starts.

The pharmaceutical composition for preventing or treating an inflammatory disease of the present invention may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents, etc., tablets, capsules, powders, It may be formulated into granules, suspensions, emulsions, syrups, and other liquids.

Specifically, the pharmaceutical composition for preventing or treating an inflammatory disease of the present invention may be formulated for oral administration, for example, tablets, troches, lozenges, water-soluble or oily suspensions, prepared powders or granules, emulsions. , Hard or soft capsules, syrups or elixirs. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for preparation in formulations such as tablets and capsules; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. Capsules contain liquid carriers, such as fatty oils, in addition to the substances mentioned above.

In addition, the pharmaceutical composition of the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. To formulate into a formulation for parenteral administration, the extract or subject composition is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated in unit dosage forms of ampoules or vials.

As a result of toxicity test, the pharmaceutical composition of the present invention was found to have little toxicity when orally administered to rats at a dose of 25 ml / kg, and did not show any side effects on the function of organs including liver.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1> Preparation of liquid

To a mixture of 25 g of oak, 25 g of gallweed, 25 g of dew, 22.5 g of ogapi and 2.5 g of licorice, 1,000 ml of water was poured, decocted for about 2 hours, and concentrated to about 200 ml to prepare a liquid.

<Example 2> Preparation of the subject 1

12,000 ml of water was poured into a mixture of 0.75 kg of young oak, 0.75 kg of gallweed, 0.75 kg of wort, 0.675 kg of Ogapi and 0.75 kg of licorice, and weighed for about 2.5 hours and concentrated to about 8,000 ml to obtain an extract. Separately, the mixture was gradually mixed with steamed rice made from 4 kg of rice, 1 kg of traditional yeast, and 0.05 kg of jujube. The extract was poured and fermented at 10-25 ° C. for about 3 days. The fermentation broth was filtered to separate the liquid and solid portions, heated to 55-60 ° C., and purified water was added to the filtrate to adjust the amount to 10,000 ml.

<Example 3> Preparation of the subject 2

To a mixture of 0.75 kg of young oak, 0.75 kg of gallweed, 0.75 kg of wort, 0.675 kg of cucumbers and 0.075 kg of licorice, add a homogeneous mixture of 4 kg of rice, 1 kg of yeast, yeast, and 0.05 kg of seedlings, and mix them together and add about 18,000 water. After addition of ml, fermentation was carried out at 10-25 ° C. for about 5 days. The fermentation broth was filtered to separate the liquid and solid portions, heated to 55-60 ° C., and purified water was added to the filtrate to adjust 10.000 ml to prepare a subject.

Example 4 Preparation of Alcohol Extraction Formulation

70%, 2,000 ml of ethanol was added to a mixture of 75 g of young oak, 75 g of gallweed, 75 g of wort, 67.5 g of ogapi and 7.5 g of licorice and extracted for about 6 hours to prepare an alcohol extraction formulation.

Example 5 Preparation of Other Formulations

The preparations obtained in the examples above were generally evaporated to dryness or concentrated or mixed with other auxiliaries according to the known conventional methods to prepare pills, granules, sprays, tablets and capsules, respectively.

Experimental Example 1 Toxicity and Side Effects Confirmation Test

1 times (2.5 mL / kg), 5 times (12.5 mL / kg) and 10 times the dose ratio (1.5-2.5 mL dose / kg body weight) to administer the liquid preparation prepared in Example 1 25 ml / kg) was orally administered to 30 adult rats for 90 days and observed daily for at least 30 days. As a result, no abnormalities were found in all experimental groups as well as in physiological abnormalities. Thus, the present inventors have studied the appropriate dosage and administration method of the liquid formulation for adult male and female volunteers after 20 years of age, and after determining the standard dose of the solution was administered according to the age of the patient. No other serious local or systemic side effects were identified except for gastrointestinal discomfort, transient manifestations or alcoholic incidence in a small number of people administered, and gastrointestinal discomfort was reduced by 1/2 to 2/3. After that, it disappeared.

Example 2 Assay of Cytokine Content in Human Mast Cells

The causes of inflammatory neuropathy disease are various, and the direction of the study is progressing from various angles.

Among them, the investigation of cytokine secretion in mast cells is a good indicator for studying such diseases. Among the cytokines secreted by mast cells, TNF-α, IL-1 and IL-6 are inflammatory cytokines. Therefore, when an effective drug inhibits the expression of these cytokines, it may release the anti-inflammatory effect of the drug. Accordingly, the present inventors treated the alcohol extracting agent of the present invention to HMC-1 cell line, which is a human mast cell line purchased from the National Institutes of Health, and then related to the secretion of TNF-α, IL-1 and IL-6 from the cells. Was investigated.

Specifically, the present inventors found that the liquid preparation prepared in Example 1, the subject preparation prepared in Examples 2 and 3, and the alcohol extracting preparation prepared in Example 4 were produced by stimulation of IgE-Ag with interleukin. In order to determine if the secretion of is controlled, the solution or subject was pretreated with the cells prior to stimulating the cells with the antigen. To examine the concentrations of TNF-α, IL-1 and IL-6 in the cultured supernatant, ELISA was performed in duplicate in 96 well ELISA plates.

Monoclonal antibodies against cytokines, purified anti-murine TNF-α and IL-1 and IL-6 were diluted with PBS (pH 7.4) to wash 96-well plates with PBS containing 0.05% Tween 1 Blocking for 1 hour with PBS containing% BSA, 5% sucrose and 0.05% NaN ₃ . After washing several times, the supernatant obtained by centrifugation and standard cytokine were added and left at 37 ° C. for 2 hours. The wells were washed and then rabbit IgG-conjugated alkaline phosphatase and avidin peroxidase were added and left at 37 ° C. for 20 minutes. After the wells were washed again, PNPP and ABTS substrate were added and the color reaction was measured at 405 nm using an ELISA reader.

As a result, when the subjects of the present invention were treated with mast cells, the TNF-α and IL-6 secretion showed almost 100% inhibition at the sample concentration of 0.1 mg / ml, and the IL-1 secretion was about 44%. Inhibition rate was shown ( Table 1 ).

On the other hand, the compositions prepared in Examples 1, 3 and 4 was lower than the effect of the composition of Example 2, but also showed an inhibitory effect on mast cell secretory cytokines. These results showed that the secretion of TNF-α, IL-6 and IL-1 was induced by the liquid preparation prepared in Example 1, the subjects prepared in Examples 2 and 3, and the alcohol extraction preparations prepared in Example 4 in mast cells. To adjust.

From the above results, patients with chronic inflammatory reactions including inflammatory neuropathy disease by controlling the three cytokines involved in the inflammatory response by the mechanism of the present invention inhibit the expression of TNF-α, IL-6 and IL-1 It was confirmed that the composition is useful for the prevention or treatment of.

Experimental Example 3 Measurement of Cytokine Expression in Peripheral Blood Mononuclear Cells (PBMC) Isolated from Human Peripheral Blood

The present inventors measured the effect of the subject composition of the present invention prepared in Example 2 on the expression of cytokines in PBMCs isolated from mast cell lines and human peripheral blood.

<3-1> Screening of objects and separation of PBMC

We collected peripheral blood from three patients with active rheumatoid arthritis (two women and one man). All selected patients with rheumatoid arthritis (mean age 61.0 ± 7.5 years; mean duration of disease 7.7 ± 4.5 years) met the criteria of the American Rheumatism Association (Arnett FC et al., J. Korean Soc. Food). Nutr ., 1996 , 25, 170-179). To investigate the specific effects of the subject compositions of the present invention on rheumatoid arthritis, we selected three healthy humans and three non-arthritic patients of similar age as controls and from which peripheral blood was drawn. Three patients who were not associated with rheumatoid arthritis were selected as controls of the disease, which were liver cirrhosis patients, cerebral infarction patients and post-cerebral infarction patients, respectively. Peripheral blood was collected into a tube containing EDTA. In order to minimize the in vitro effect on the activation phase of the cells, monocytes were immediately isolated by rapid centrifugation (Ficoll-Hypaque, Histopaque-1077) at room temperature. PBMCs were collected from the interface layer and washed twice with PBS and once more with RPMI 1640 medium. PBMC was stabilized by incubating for 2 hours in a 5% CO ₂ incubator at 37 ℃ in RPMI-1640 medium. Stabilized cells at a concentration of 6 × 10 ⁵ cells / ml were seeded into the culture plates.

<3-2> Cell Culture and Stimulation

HMC-1 cell line, a human leukemia mast cell line, contains 100 units / ml penicillin, 100 µg / ml streptomycin, 10 ^-5 M monothioglycerol and 10% fetal bovine serum inactivated by heat. ) Was incubated at 37 ° C., 5% CO ₂ in IMDM (Iscove's modified Dulbecco's medium, Gibco BRL) medium. Concentration of 1-100 μg / ml 30 minutes before stimulating HMC-1 cells at 1 × 10 ⁶ cells / ml using 20 nM of PMA (phorbol 12-myristate 13-acetate, Sigma) or 1 μM of A23187 The subject composition prepared in Example 2 of the invention was treated and then incubated at 37 ° C. Human PBMCs were cultured in RPMI-1640 medium supplemented with 100 units / ml penicillin, 100 μg / ml streptomycin and 10% FBS inactivated by heat. PBMCs at a concentration of 6 × 10 ⁵ cells / ml were pretreated with the subject composition at a concentration of 0.1-10 μg / ml, and then incubated for 24 hours after stimulation with 25 μg / ml of PHA (phytohaemagglutinin, Sigma).

<3-3> MTT analysis

To investigate the viability of the cells, we performed MTT colorimetric analysis according to the methods of the prior art (Kim MS et al., Cancer Lett ., 2001 , 171, 75-89). Briefly, 5 × 10 ⁴ cells / ml of HMC-1 cells were cultured for 8 hours after stimulation with PMA or A23187 in the presence or absence of the subject composition of the present invention. After the addition of the MTT solution, the cells were incubated at 37 ° C. for 4 hours, and the absorbance at 540 nm was measured after lysis of the crystallized MTT.

The results of the experiments were expressed as mean ± SEM for the indicated number of experiments. The difference between the PMA or A23187 treated group and the control group and the significant degree between the PMA or A23187 treated group and the subject composition treated group of the present invention was determined by the Mann-Whitney U test. Differences between the rheumatoid arthritis group, disease control group and healthy control group were compared by variance analysis (ANOVA) taking into account the post-hoc test of the mean according to Tukey's method. In all tests, it was considered meaningful if it had a P value of 0.05 or less.

As a result, the subject composition of the present invention did not show a toxic effect on the viability of HMC-1 cells. 1 shows the viability of cells cultured for 8 hours after stimulation with 20 nM PMA or 1 μM A23187 in the presence or absence of the subject composition at a concentration of 100 μg / ml. In the cells treated with PMA or A23187, the viability of the cells was slightly decreased by 87.5 ± 7.0% and 91.4 ± 1.6% when compared with the control group 100 ± 4.0%. However, the subject composition of the present invention did not affect the viability of the cells in each condition and did not show toxicity to HMC-1 cells.

<3-4> Measurement of effect on secretion of cytokine

We measured the concentration of secreted cytokines present in the culture supernatants of HMC-1 or PBMC by sandwich ELISA methods according to the manufacturer's instructions (R & D Systems, IL for TNF-α and IL-1β assays). -8 PharMingen for analysis). Absorbance of the avidin-horseradish peroxidase color reaction was measured at 405 nm and compared with the absorbance measured using serial dilution of a standard human recombinant. It was.

As a result, the subject composition of the present invention inhibited TNF-α, IL-1β and IL-8 secretion in HMC-1 cells. HMC-1 cells are known to be activated by non-physiological agents such as PMA or A23187 to release various types of cytokines (Queralt M, et al., Inflamm. Res ., 2000 , 49, 355-360; Kruger-Krasagakes S, Immunol ., 1999 , 98, 253-257). In preliminary experiments, we investigated the optimal time conditions for secreting the pro-inflammatory cytokines TNF-α, IL-1β and IL-8 by PMA or A23187. The secretion of TNF-α reached its maximum when cultured for 8 hours after 20 nM PMA stimulation ( A in FIG. 2 ). Compared with no stimulation, TNF-α increased more than 10-fold in the supernatant after stimulation for about 8 hours. We investigated the inhibitory effect of the subject compositions of the invention on TNF-α excretion in PMC-induced HMC-1 cells ( FIG . 2B ). The subject composition of the present invention inhibited the secretion of TNF-α in a dose-dependent manner and almost completely inhibited the secretion of TNF-α when treated with a 100 μg / ml subject composition ( 87% inhibition). Figure 3 A is a IL-1β release time in the HMC-1 cells stimulated with a 1 μ M A23187 - shows the response (time-response) curves. The amount of IL-1β released into the supernatant after 6 hours of stimulation was nearly threefold higher than in the absence of stimulation. The effect of the subject composition of the present invention for the secretion of IL-1β in HMC-1 cells are shown in B of 3. The subject composition of the present invention inhibited the secretion of IL-1β in a DOS-dependent manner and provided only statistically significant levels when treated with 10 μg / ml of the subject composition. As shown in A of FIG . 4 , we determined the time at which IL-8 secretion reached its maximum after stimulation with 1 μM of A23187. The secretion of IL-8 reached the maximum value when cultured for 6 hours after stimulation, and increased by 15-fold in the supernatant compared to the case without stimulation. The effect of the subject composition of the present invention on IL-8 secretion is shown in B of FIG. 4 . The subject composition of the present invention inhibited the secretion of IL-8 in a DOS-dependent manner, with 84% inhibition when treated with 100 μg / ml of the subject composition.

<3-5> RNA isolation and RT-PCR analysis

We extracted mRNA from the collected cells using a micro mRNA purification kit (Amersham Pharmacia Biothch), using a first strand cDNA synthesis kit (Amersham Pharmacia Biotech) at 37 ℃ 1 hour 30 100 ng and mRNA were converted to cDNA by reverse transcriptase reaction for minutes. PCR amplification was repeated 35 times 45 seconds at 94 ° C., 45 seconds at 60 ° C. and 1.5 minutes at 72 ° C. using a set of commercially available oligonucleotide primers for human TNF-α, IL-8 and β-actin It was performed by. Primer sequences for IL-1β were obtained from published data by Yamamura et al. (Yamamura M, et al., Science , 1991 , 254, 277-279) and samples were 1 min at 94 ° C., 1 min and 72 ° C. Amplification was repeated 35 times for 1 minute at &lt; RTI ID = 0.0 &gt; The primer set was used to produce PCR products of 355, 248, 200 and 661 bp lengths for TNF-α, IL-1β, IL-8 and β-actin, respectively. The final PCR product was subjected to electrophoresis on 2% agarose gel.

As a result, the subject composition of the present invention inhibited the expression of TNF-α and IL-1β in HMC-1 cells but did not inhibit the expression of IL-8. To determine whether the subject compositions of the invention can regulate the expression of cytokine mRNA, we isolated mRNA from HMC-1 cells and reverse transcribed them into cDNA. cDNA was amplified by PCR using primers specific for TNF-α, IL-1β and IL-8 and finally produced bands of 355, 248 and 200 bp, respectively, on 2% agarose gels ( FIG. 5 ). . PCR using the structural gene human β-actin (661 bp) was performed to confirm the equivalence of cDNA. When incubated for 7 hours in the presence of PMA, the expression of TNF-α was increased compared to that without PMA treatment, and the subject composition of the present invention inhibited the expression of TNF-α in a concentration-dependent manner ( A ) of FIG. 5 . Higher IL-1β expression was induced in cells stimulated with A23187, and pretreatment with 10 μg / ml of the subject composition successfully inhibited IL-1β expression, consistent with data from ELISA ( FIG. 5 ) . B ). When cultured for 5 hours after treatment with A23187, IL-8 expression was increased compared to the case without treatment. However, the subject composition did not inhibit IL-8 expression in cells stimulated by A23187 ( FIG . 5C ).

Comparison of Moderating Effects on Cytokine Secretion in Rheumatoid Arthritis Patients and Control Subjects

In order to investigate the specificity of the subject compositions of the invention for active rheumatoid patients, we compared the production of cytokines by PBMCs isolated from healthy or non-rheumatic patient controls of the same age.

As a result, as shown in Table 1, production of TNF-α and IL-1β was increased by PHA-stimulation in PBMC (Mao TK, et al., Life Science , 2000 , 66, 1377-1386; Mayringer I, et al., J. Immunol.Methods , 2000 , 235, 33-40). Compared to unstimulated PBMCs, production of TNF-α increased 4.5-fold for active rheumatoid patients, 3.1-fold for unhealthy controls and 5.9-fold for healthy controls (p <0.05). Pretreatment of the subject composition of the present invention at 10 μg / ml slightly inhibited the production of TNF-α in the healthy control group (p = 0.158), but the dose-dependent method of TNF-α in the active patient group. Production was inhibited. In the active rheumatoid arthritis group, PHA treatment resulted in a 3.9-fold increase in IL-1β production by PBMC, a 3.7-fold increase in the unhealthy control group, and a 12.7-fold increase in the healthy control group (0 < 0.05). Treatment of the subject composition of the present invention 30 minutes prior to stimulation with PHA inhibited the production of IL-1β in a DOS-dependent manner in PBMCs isolated from the active rheumatoid arthritis group, but not in the control group. .

As described above, the composition of the present invention can be usefully used as a prophylactic or therapeutic agent for inflammatory neuropathy diseases such as degenerative arthritis, rheumatoid arthritis, neural arthritis due to physical damage, inflammatory low back pain and the like. In particular, the composition of the present invention is very excellent in the treatment effect as well as alleviate the symptoms of degenerative arthritis, which occurs frequently after the elderly.

Claims (13)

Water extracts or alcohol extracts of a mixture of cypress, oleander, dew, cucumber, and licorice for the prevention or treatment of inflammatory diseases. The water extract or alcohol extract of claim 1, wherein the alcohol is selected from the group consisting of 10 to 100% of methanol, ethanol, propanol and butanol. The water extract or alcohol extract of claim 2, wherein the alcohol is 70 to 99.9% ethanol. The method according to any one of claims 1 to 3, characterized in that the oak, gallweed, hyssop, cucumber and licorice are mixed in a weight ratio of 0.5 to 1: 0.5 to 1: 0.5 to 1: 0.1 to 1: 0.05 to 0.1. Water extract or alcohol extract. The subject composition for the prophylaxis or treatment of an inflammatory disease prepared by adding fermentation to a water extract or an alcohol extract of claim 1, wherein the mixture of rice, yeast or yeast is added to the mixture. 6. The main composition according to claim 5, wherein the rice, yeast and chimney are mixed in a weight ratio of 1 to 4: 0.2 to 1: 0.02 to 0.1. 1) heating by adding water or alcohol to a mixture of cypress, gallweed, dew, organza and licorice; And 2) A method for producing an extract for preventing or treating an inflammatory disease of claim 1, comprising separating the liquid portion and the solid portion and then concentrating the liquid portion. 8. The production method according to claim 7, wherein the oak, gallweed, hyssop, cucumber, and licorice are mixed in a weight ratio of 0.5 to 1: 0.5 to 1: 0.5 to 1: 0.1 to 1: 0.05 to 0.1. 1) fermenting the water extract or alcohol extract by adding a mixture of rice, yeast or strains separated from the yeast and the mother seed; And 2) A method of preparing a subject composition for preventing or treating an inflammatory disease according to claim 5, wherein the liquid portion and the solid portion are separated and the liquid portion is concentrated. 10. The process according to claim 9, wherein the rice, yeast, and chimney are mixed in a weight ratio of 1 to 4: 0.2 to 1: 0.02 to 0.1. A pharmaceutical composition for preventing or treating an inflammatory disease, comprising the water extract or alcohol extract of claim 1 or the subject composition of claim 5 as an active ingredient. 12. The composition according to claim 11, wherein the inflammatory disease is an inflammatory neurological disease. 13. The composition of claim 12, wherein the inflammatory neurological disease is selected from the group consisting of degenerative arthritis, rheumatoid arthritis, neuroarthritis due to physical injury and inflammatory low back pain.

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