WO2007126259A1

WO2007126259A1 - Compositions for treating autoimmune disease containing extracts of sophora flavescens

Info

Publication number: WO2007126259A1
Application number: PCT/KR2007/002074
Authority: WO
Inventors: Hyun Su Bae; Min Kyu Shin; Moo Chang Hong; Moon Kyu Kang; Hwan Suck Chung; Eun Jung Ko; Nam Hee Park; Young Seop Lee; Young Nam Yim; Dong Woo Kim
Original assignee: Purimed Co., Ltd.
Priority date: 2006-04-28
Filing date: 2007-04-27
Publication date: 2007-11-08
Also published as: KR100804517B1; KR20070106336A

Abstract

Disclosed is a pharmaceutical composition comprising a Sophora flavescens extract as an active ingredient useful in the treatment of autoimmune diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis, multiple sclerosis, immune mediated or type 1 diabetes mellitus, inflammatory bowel disease, and scleroderma. Also, the Sophora flavescens extract can be used in a food composition which is thus applicable for the treatment of such autoimmune diseases.

Description

COMPOSITIONS FOR TREATING AUTOIMMUNE DISEASE CONTAINING EXTRACTS OF SOPHORA FLAVESCENS

Technical Field

The present invention relates to a composition comprising an extract from Sophora flavescens, useful in the treatment of autoimmune diseases.

Background Art

An autoimmune disease is a condition in which the body perceives its own tissues as foreign and directs an immune response against them. The treatment of autoimmune diseases, thus, requires the suppression of the body's immune system, which is likely to cause cancer or infectious diseases. Most autoimmune diseases are established over a long period of time, exhibit chronic symptoms and incur permanent injury of organs. As a matter of fact, there are almost no methods for curing autoimmune diseases. Although there have been great advances in knowledge of autoimmune diseases in the past two to three decades, pathogenesis mechanisms of autoimmune diseases, identity of auto-antigens, and regulatory genes still remain unclear. Autoimmune diseases are largely classified into organ-specific diseases and systemic diseases.

Organ-specific autoimmune diseases are induced upon immune responses against organ-specific antigens and may be generated in almost all organs. Systemic autoimmune diseases are attributed to immune responses to antigens expressed throughout the body, rather than immune responses directed against specific types of cells. Systemic autoimmune diseases may also occur selectively in specific organs (see, http://irc.ulsan.ac.kr/immunity/index.htm). Examples of autoimmune diseases include i) rheumatoid arthritis, caused when the immune system attacks various connective tissues, and ii) multiple sclerosis (MS), a demyelinating disease that affects the central nervous system. Persons with MS live relatively normally in most cases, but MS will cause blindness, paralysis, or premature death in more severe cases. Examples further include iii) immune-mediated or type I diabetes mellitus, in which immune cells destroy pancreas beta cells secreting insulin and for which the MHC represents the most important susceptibility locus, iv) inflammatory bowel disease, caused when the immune system attacks the intestine, v) scleroderma, which induces the thickening of the skin or vessels, vi) systemic lupus erythematosus (SLE) , a chronic autoimmune disease that is accompanied by severe fatigue, fever, joint pains, etc. and can affect any part of the body, but most often harms the kidneys, the brain, the lungs, etc.

(http: //home. inje. ac. kr/~lecture/immunobiotech/ch6/6autoimin unity.htm) .

Because its symptoms vary so widely, SLE often resembles, and is mistaken for, other illnesses. Thus, it is necessary to distinguish SLE from other illnesses and infectious diseases, such as HIV, malignant cancers, rheumatoid arthritis, vasculitis, and connective tissue diseases, such as mixed connective tissue disease (MCTD) . For the diagnosis of SLE, testing with antibodies, such as anti-nuclear antibody testing and anti-Sm antibody testing is highly specific.

In addition, complement assays with C3 and C4 are used for diagnosis. Patients with SLE show the symptoms and signs of mild normocytic or normochromic anemia or chronic hypochromic anemia, leukopenia, lymphocytopenia, and thrombocytopenia.

In combination with measurements of high serum anti- nuclear antibody and anti-dsDNA antibody levels and low serum complement titers, the detection of albuminuria, hematuria, cellular casts, and granular casts together with an increase in urinary protein level leads to the diagnosis that SLE and lupus nephritis is of high activity.

With regard to renal manifestation, most SLE patients (90-100%) are found to develop renal impairment with lupus nephritis. Drugs may induce or aggravate SLE. However, symptoms of drug-induced lupus generally disappear once a patient is taken off the medication that triggered the episode. There is a lot of medication currently in use that can cause this condition, though the most common drugs are hydralazine, sulfonamide, penicillin, anticonvulsants, tetracycline, and procainamide .

As SLE is a chronic disease with no known cure, treatment is restricted to increasing the quality of life and lifespan of the patients.

Lupus is treatable symptomatically, mainly with corticosteroids, immunosuppressants, and cytotoxic agents, though there is currently no cure. Recently, plasma change is emerging as a new therapy for SLE. However, these therapies are also accompanied with various side effects, such as Cushing's syndrome, malignant tumor, hypertension, osteonecrosis, osteoporosis, psychopathy, ovarian failure, and hepatotoxicity.

Extracts from Sophora flavescens, mainly from Sophorae radix, have been used as a stomachics, a vasoconstrictor, an antipyretic analgesic and a dermaparasiticide. Major ingredients found in Sophora flavescens are alkaloids, such as matrine and oxymatrine, and flavonoids, such as kushenol H, kushenol K, kurarinol and kuraridine. In herb medicine, extracts from Sophora flavescens are used in the treatment of various diseases including dyspepsia, neuralgia, hepatitis, tonsillitis, pneumonia, and jaundice. It is described in ancient herb medicinal literature that when applied to the body, Sophora flavescens shows heat and humidity controlling activity, stroke resistance, parasiticidal activity, and diuresis. In fact, Sophora flavescens is reported to have antiinflammatory activity, immunity enhancement, anti-bacterial activity and leukocyte promotion. Nowhere are extracts from or ingredients of Sophora flavescens mentioned with regard to use in autoimmune diseases in previous literature.

Leading to the present invention, intensive and thorough research into the medicinal effects of Sophora flavescens, conducted by the present inventors, resulted in the finding that Sophora flavescens extracts, as tested in NZB/W Fl (New Zealand Black/White Fl) mice, induced a decrease in albuminuria, total anti-dsDNA IgG, and IFN-γ while increasing lymphocytes and IL-4, and that the mice showed neither injured glomeruli nor macrophage infiltration. It was concluded that Sophora flavescens extracts are effective in the treatment of autoimmune diseases.

Disclosure of the Invention

It is therefore an object of the present invention to provide a pharmaceutical composition for the treatment of autoimmune diseases, comprising an extract from Sophora flavescens as an active ingredient.

Other objects and features of the present invention will be more apparent in the following in the following detailed description, claims and description of drawings.

Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a histogram showing the body weight of the NZB/W Fl mice administered with a Sophora flavescens extract, increased in comparison to that of a control;

FIG. 2 is a histogram showing the albuminuria level of NZB/W Fl mice administered with a Sophora flavescens extract, decreased in comparison to that of a control administered with distilled water;

FIG. 3 is a histogram showing the effect of Sophora flavescens on GOT and GPT activity in NZB/W Fl mice; FIG. 4 is a histogram showing total protein levels of control NZB/W Fl mice and Sophora flavescens extract- administered mice;

FIG. 5 is a histogram showing creatinine levels of control NZB/W Fl mice and Sophora flavescens extract- administered mice;

FIG. β is a histogram showing the BUN (blood urea nitrogen) level of NZB/W Fl mice administered with the Sophora flavescens extract, decreased in comparison to that of a control;

FIG. 7 is a histogram showing the anti-dsDNA total IgG level of NZB/W Fl mice administered with a Sophora flavescens extract, decreased in comparison with that of a control; FIG. 8 is a histogram showing the anti-dsDNA IgG subclass (IgGl) level of NZB/W Fl mice administered with a Sophora flavescens extract, decreased in comparison with that of a control;

FIG. 9 is a histogram showing the anti-dsDNA IgG subclass (IgG2a) level of NZB/W Fl mice administered with a Sophora flavescens extract, decreased in comparison with that of a control;

FIG. 10 is histograms showing the effect of the

Sophora flavescens extract on the expression of IFN-γ and IL-4 in cell culture media of spleen lymphocytes from control mice and Sophora flavescens extract-administered

NZB/W Fl mice; and

FIG. is histopathological photographs, 400-fold magnified, showing glomerulosclerosis (short wide arrow) and macrophage infiltration (long arrow) in control (panel A) and relatively very low glomerulosclerosis and no macrophage infiltration around vessels in Sophora flavescens extract-administered mice (panel B) .

Best Mode for Carrying Out the Invention

In accordance with a first aspect thereof, the present invention is directed to a pharmaceutical composition for the treatment of autoimmune diseases, comprising a Sophora flavescens extract as an active ingredient, wherein the autoimmune diseases are selected from among systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, immune-mediated or type 1 diabetes mellitus, inflammatory bowel diseases, scleroderma, and combinations thereof.

Rheumatoid arthritis is traditionally considered a chronic, inflammatory autoimmune disorder that causes the immune system to attack the joints, with the concomitant symptoms of symmetric joint inflammation, joint pains, swelling, and ankylosis. Osteoarthritis, a kind of rheumatoid arthritis, also known as degenerative arthritis, is a condition which is caused by cartilage wear.

Multiple sclerosis is a chronic, inflammatory, demyelinating disease that affects the central nervous system, and is triggered by T cells. MS allows patients to live relatively normally in most cases, but can cause severe symptoms, including blindness, paralysis or premature death.

Immune-mediated or type 1 diabetes mellitus is an autoimmune disease that results in the permanent destruction of insulin producing beta cells of the pancreas . The lack of insulin leads to hyperglycemia with various complications (fatigue, frequent urination, thirst, hyperosmolar coma) . In severe cases, diabetes causes renal failure, blindness, limb loss, and death. Inflammatory bowel diseases refer to chronic diseases in which the immune system attacks the intestines, with concomitant symptoms of diarrhea, emesis, vertigo, abdominal convulsions, and pain.

Scleroderma may lead to the thickening of the skin or vessels. Its complications include discomfort in muscle and shortness of breath and, in severe cases, failure of the heart, the kidneys, the lungs and the intestine.

In addition to the active ingredient, the pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. As long as it is typically used in the formulation of pharmaceutical compositions, any carrier is useful in the present invention. Examples of carriers useful in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. The pharmaceutical composition of the present invention may further comprise a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspension agent, a preservative, etc. Pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995) .

The pharmaceutical composition of the present invention may be administered orally or parenterally. Parental administration may be conducted by intravenous, subcutaneous, muscular, abdominal or transdermal injection. Depending on various factors, including formulation method, administration type, patient's age, body weight, sex, severity of disease, diet, administration frequency, administration route, excretion rate, and response sensitivity, the dosage of the active ingredient may vary. Physicians skilled in the art can easily determine and prescribe a therapeutically effective amount of the pharmaceutical composition.

According to techniques known to those skilled in the art, the pharmaceutical composition of the present invention may be formulated with pharmaceutically acceptable carriers and/or vehicles into unit dosage forms or multiple dosage containers. The formulation may be in the form of solutions in oil or aqueous matrices, suspensions, emulsions, elixirs, powders, granules, tablets, or capsules. It may further comprise a dispersant or a stabilizer.

In accordance with a second aspect thereof, the present invention is directed to a food composition useful in the treatment of autoimmune diseases, comprising an extract from Sophora flavescens. In an embodiment of this aspect, the; autoimmune diseases are systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, immune mediated or type 1 diabetes mellitus, inflammatory bowel disease and scleroderma.

In addition to the Sophora flavescens extract, the food composition of the present invention may comprise ingredients which are usually used in the art, such as proteins, carbohydrates, lipids, nutrients, seasonings, etc. For example, when the food composition of the present invention is used for beverages, it may further comprise citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, a Eucowmia ulmoides olivon extract, a jujube fruit extract, and/or a licorice extract. When considering its ready availability, the food composition of the present invention is very useful in the treatment and prevention of neurological illness or oxidative stress-caused diseases and the amelioration of cognitive function.

NZB/NZW Fl mice can produce a predetermined portion of auto antibodies, and are typically used as a study model for human SLE. Although born normally, NZB/NZW mice suffer from hemolytic anemia for 2-3 months after birth, with positive responses detected in all assays for anti- erythrocytic antibody, antinuclear antibody, lupus cell, and immune complex.

GOT (AST) and GPT (ALT), both involved in the synthesis of amino acids, are located mainly in the liver. These enzymes are maintained at constant levels in blood by normal cell disruption, but the levels increase as the enzymes are released from the cells upon cell disruption in the event of injury of the liver or organs. Serum total protein refers to the total amount of protein in the blood, indicating the nutrient state of the body. Serum total protein increases slightly upon blood concentration by dehydration, with no clinical significance imposed thereto. A sharp increase in serum total protein is found in multiple myeloma, whereas concentrations below a normal reference range reflect malnutrition, chronic infection, liver cirrhosis, nephrotic syndrome, and chronic inflammation. Creatinine is a breakdown product of creatine phosphate in muscle, and is mainly filtered by the kidney, though a small amount is actively secreted. Kidney diseases, such as renal failure, nephritis, etc. thus increase the level of creatinine in blood. Also, an increase in creatinine serum level is detected in association with high protein diets, tissue destruction and gastrointestinal bleeding as well as muscular diseases. Liver diseases, such as liver cirrhosis, slightly decrease the creatinine level the in blood, but no clinical significance is attached thereto. BUN (blood urea nitrogen) increases in serum level, along with creatinine, in the event of kidney diseases, and decreases in the event of liver diseases such as liver cirrhosis, but no clinical significance is assigned thereto. High protein meals, tissue destruction, gastrointestinal bleeding and dehydration also increase serum BUN levels. Thus, there is a need to distinguish between them. Interferons (IFN) are classified largely into IFN-α, IFN-β and IFN-γ. IFN-γ is produced mainly by activated T cells. The binding of interferons to their receptors elicits various effects. Particularly, IFN-γ, secreted by T lymphocytes and NK cells, when associated with its receptor, mediates immunity enhancement and the activation of antigen-presenting cells, that is, macrophages. Another important function of IFN-γ is to induce MHC class II expression. Other interferons increases the expression of the MHC class I gene. IL-4 is suggested as a therapeutic for ThI response-induced autoimmune diseases (type 1 diabetes mellitus, rheumatoid arthritis, multiple sclerosis) because it acts as an antagonist against IFN-γ, produced by ThI cells which promote cell-mediated immunity. Found to suppress the growth of malignant tumors (plasmacytoma, mammary adenocarcinoma, transformed fibroblast cell lines, a melanoma cell line, sarcoma cell line, etc.), IL-4 is also clinically tested for effectiveness and safety in use as a novel anticancer agent on levels I/II.

After the oral administration of the Sophora flavescens extract according to the present invention to NZB/W Fl mice, albuminuria was significantly decreased, showing a level of 2.38±0.36 in a Sophora flavescens- administered group compared to 3.67±0.31 in a control group. There is immunoglobulin deposit in the glomeruli of most SLE patients, which is the main cause of lupus nephritis. Almost half of SLE patients show an albuminuria manifestation. Urine analysis is one of the most simple and frequently used tests for lupus nephritis. A decrease in albuminuria is believed to result from the amelioration of lupus.

The Sophora flavescens extract according to the present invention was found to activate lymphocytes . Recently, autogenous hematopoietic stem cells have been studied for the treatment of lupus diseases. After the complete removal of ill lymphocytes, they are substituted with normal lymphocytes freshly produced from implanted autogenous hematopoietic stem cells. The Sophora flavescens extract according to the present invention was observed to increase the amount of lymphocytes, but it has not yet clear that they were ill or normal lymphocytes. However, the Sophora flavescens extract according to the present invention reduced the levels of monocytes, neutrophils and eosinophils, requiring additional study on the relationship between lupus and lymphocyte increase/decrease.

It was also found that the Sophora flavescens extract according to the present invention induced a reduction in the level of anti-dsDNA total IgG, which is a criterion for lupus diseases. Anti-dsDNA antibodies play a decisive role in the diagnosis of lupus because they are detected only in patients with lupus. The total level of anti-dsDNA antibodies is widely used as an index of lupus treatment because it changes in direct proportion with the condition of lupus, that is, increases with the deterioration of lupus and decreases with the amelioration of lupus. Therefore, the Sophora flavescens extract of the present invention is useful in the treatment of lupus diseases.

Further, the Sophora flavescens extract according to the present invention was found to deactivate IFN-γ. IFN-γ is known to improve the condition of patients with rheumatoid arthritis, but to deteriorate the condition of patients or mice suffering from SLE. IFN-γ functions to promote the production of IgG2a, resulting in glomerular disorders. IL-4, a cytokine produced by Th2, induces the secretion of IgGl and IgE, and an increased level thereof is detected in SLE patients. In the assay for effect on cytokine expression in spleen lymphocytes of NZB/W Fl mice, the Sophora flavescens extract of the present invention was determined to be effective in the treatment of lupus diseases as a Sophora flavescens extract-administered group was significantly decreased in the level of IFN-γ, involved in IgG2a conversion, compared to a control group. In contrast, an increase in IL-4, involved in IgGl conversion, was detected in a Sophora flavescens extract-administered group, compared to a control group, but no significance was attached thereto.

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

In the following experiments, measurements are expressed as mean ± SEM (standard error of mean) and a Student's t-test was conducted for statistical analysis. *, p<0.05 (n=8) .

<EXAMPLE 1> Effect of Sophora flavescens Extract on Body Weight of NZB/W Fl Mice

(1) Preparation of Sophora flavescens extract and Administration method

A Sophora flavescens extract, purchased from Sun Ten

Pharmaceutical co. Ltd. (Lot No. 102401, Taipei, Taiwan), was diluted and dissolved in water for 24 hours to give a 1% solution. After centrifugation at 3,000 rpm for 10 min, the supernatant was filtered through filter paper (Whatman,

No.2, Portland, OR, USA). The filtrate, a 1% Sophora flavescens extract solution, was orally administered to

NZB/W Fl mice every day in a dose of 6 ml a day per mouse for 15 weeks. Yield: (Sophora flavescens 0.68g + starch

0.32g)/lg = 68%

(2) Preparation of NZB/W Fl mice

Six-week-old NZB/W Fl (New Zealand Black/White Fl) female mice purchased from Central Lab. Animal Inc. (Seoul, Korea) were used as experimental animals in the present invention. They were fed with irradiated foods commercially available from Purina (Kyung-ki-do, Korea) and sterile distilled water. The NZB/W Fl mice were allowed to freely approach the feed and water under general light conditions at a constant temperature of 22+1°C and a constant humidity of 55+5% for 3 months.

The mice were divided into two groups of 8 each: a control administered with feed and distilled water and a test group administered with the 1% Sophora flavescens extract solution.

(3) Effect of Sophora flavescens extract on the body weight of mice

NZB/W Fl mice three months old were orally administered with the 1% Sophora flavescens extract solution for 15 weeks before measurement of body weight on a balance (OHAUS Corporation, Pine Brook, NJ) . The body weight was measured to be 39.38+1.35g for the control and 41.13±1.32g for the test group, with no significant difference in the increase (see FIG. 1) .

<EXAMPLE 2> Decrease in Albuminuria with Administration of Sophora flavescens

In order to determine whether the Sophora flavescens extract according to the present invention causes glomerular injury leading to kidney dysfunction, the 3- month-old NZB/W Fl mice of (2) of Example 1 was orally administered with the 1% Sophora flavescens extract solution of (1) of Example 1 for 15 weeks. Urine samples taken from the mice were assayed for albuminuria using URiSCAN strips (YD Diagnostics.com) according to the manufacturer's protocol. On the basis of the data of the color table provided by the manufacturer, 1 point was given to a level of 10-29 μg/ml, 2 points to a level of 30-99 μg/ml, 3 points to a level of 100-299 μg/ml, 4 points to a level of 300-999 μg/ml, and 5 points to a level of 1000 μg/ml or larger. The test group gained 2.38±0.36, which was decreased compared to 3.67±0.31 of the control, with significance (see FIG. 2) .

<EXAMPLE 3> Effect of Sophora flavescens on Liver Function of NZB/W Fl Mice

In order to examine whether the Sophora flavescens extract causes liver injury, the 3-month-old NZB/W Fl mice of (2) of Example 1 was orally administered with the 1% Sophora flavescens extract solution of (1) of Example 1 for 15 weeks. Blood samples taken from the heart were analyzed for GOT and GPT, which are released into the blood when hepatocytes are disrupted by, for example, drug or bile duct stasis.

The blood sampled from the heart was left at room temperature for 1 hour, followed by centrifugation at 3000 rpm for 5 min. The sera thus separated were stored at -20°C and assayed for GOT and GPT using DRI-CHEM 350Os(FuJi Photo Film Co., Ltd, Kamagwa-ken, Japan).

There was no significant difference between the test group and the control (Table 1, FIG. 3)

TABLE 1 GOT and GPT in NZB/W Fl Mice Administered with Sophora flavescens Control Sophora flavescens Administered

GOT (U/l) 65 0+10 63 47 71±6. 71

GPT (ϋ/1) 20 33±5 93 18 88+1. 54

Values expressed as mean±SEM

Student's t-test was conducted for statistical analysis. *: p<0.05 (n=8)

<EXAMPLE 4> Effect of Sophora flavescens on Renal Function in NZB/W Fl Mice

In order to examine whether the Sophora flavescens extract has a negative influence on renal function, the 3- month-old NZB/W Fl mice of (2) of Example 1 were orally administered with the 1% Sophora flavescens extract solution of (1) of Example 1 for 15 weeks. Blood samples were taken from the heart. Then, the blood was left at room temperature for 1 hour, followed by centrifugation at 3000 rpm for 5 min. The sera thus separated were stored at -20°C and assayed for total protein, creatinine and BUN (blood urea nitrogen) using DRI-CHEM 3500s (Fuji Photo Film Co., Ltd, Kamagwa-ken, Japan) .

The test group was reduced with regard to all of the test items, compared to the control, but with no statistical significance (Table 2, FIGS. 4 to 6) .

TABLE 2 Change in the Level of Total Protein, Creatinine and BUN in NZB/W Fl Mice upon Administration of Sophora flavescens

Values mean±SEM

Student' s t-test was conducted for statistical analysis. *: p<0.05 (n=8)

<EXAMPLE 5> Effect of Sophora flavescens on Blood Cell Count in NZB/W Fl Mice

In order to examine the influence of the Sophora flavescens extract on the count and distribution of each blood cell, the 3-month-old NZB/W Fl mice of (2) of Example

1 was orally administered with the 1% Sophora flavescens extract solution of (1) of Example 1 for 15 weeks. Blood samples were taken from the heart and analyzed for blood cell count and distribution using a hematology analyzer.

The blood samples were placed in EDTA-treated tubes which were then rolled on a roll mixer for 30 min, followed by analysis of counts of blood cells, that is, leukocytes, erythrocytes, lymphocytes, neutrophils, monocytes, eosinophils and basophils.

The lymphocyte count of the test group was increased compared to that of the control, with significance, but there was almost no difference in the distribution of other blood cells therebetween (Table 3) .

TABLE 3

Change in Blood Cell Count of NZB/W Fl Mice upon Administration of Sophora flavescens

Control Sophora flavescens Administered

Leukocyte (K/μϋ) 1.5310.24 1.65 + 0.24

Erythrocyte (M/βi) 7.96+0.33 7.75 ± 0.33

Lymphocyte (%) 84.80±0.92 89.45 ± 1.61*

Neutrophil (%) 8.61±0.93 6.10 ± 1.99

Monocyte (%) 5.37±0.79 4.94 ± 0.42

Eosinophil (%) 0.88±0.24 1.66 ± 0.59

Basophil (%) 0.25±0.07 0.27 + 0.23

Values mean±SEM

Student's t-test was conducted for statistical analysis. *:p<0.05 (n=8)

<EXAMPLE 6> Effect of Sophora flavescens Extract on Expression of Anti-dsDNA Antibody in NZB/W Fl Mice

(1) Material and Method

The 3-month-old NZB/W Fl mice of (2) of Example 1 were orally administered with the 1% Sophora flavescens extract solution of (1) of Example 1 for 15 weeks. Blood samples taken from the heart were analyzed for the expression level of anti-IgG, IgGl and IgG2a in the NZB/W Fl mice using a mouse-IgG, IgGl, IgG2a ELISA Quantitation kit (Bethyl Laboratory, Montgomery, TX, USA) according to the manufacturer's protocol. In this regard, calf thymus DNA (Sigma, Louis, MO, USA) was diluted to a concentration of 100 μg/ml in a coating buffer (0.05M carbonate- bicarbonate, pH 9.6). The dilution was plated at an amount of 100 μl per well in 96-well plates and allowed to stand for 1 hour at room temperature. The plates thus coated with the calf thymus DNA were washed three times with a wash buffer (5OmM Tris, 0.14M NaCl, 0.05% Tween-20) and incubated with 200 μl/well of a blocking buffer (5OmM Tris, 0.14M Nacl, 1% BSA, pH 8.0) for 30 min at room temperature. After washing the plates three times with the wash buffer, blood samples were plated at an amount of 100 μl per well and incubated for reaction at room temperature for 1 hour. Thereafter, five washings with the wash buffer preceded incubation with 100 μl of HRP antibody per well for 1 hour at room temperature. Again, five washings with the wash buffer were followed by the addition of 100 μl of a substrate solution (TMB Substrate Reagent; Pharmingen, BD Bioscience, San Diego, CA, USA) per well and incubation for 30 min at room temperature in the shade. 30 min after the addition of 100 μl of 2N H₂SO₄ per well, absorbance was read at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA) .

(2) Result The test group was determined to have anti-dsDNA total IgG at an amount of 1446.01330.5 ng/ml, which was decreased compared to the amount detected in the control, 2889.0+461.6 ng/ml, with significance. The level of anti- dsDNA IgGl was measured to be 2070.01772.4 ng/ml in the control and 140.0180.1 ng/ml in the test group with a significant decrease. As for the level of anti-dsDNA IgG2a, it was also significantly decreased in the test group (475.2+138.3 ng/ml) compared to the control (864.7+33.7 ng/ml) (Table 4, FIGS. 7 to 9)

TABLE 4

Effect of Sophora flavescens on Anti-dsDNA Level in NZB/W

Fl mice

Control Sophora flavescens- Administered

Anti-dsDNA total IgG 2889 .01461 .6 1446.0 + 330.5 * (ng/ml)

Anti-dsDNA IgGl (ng/ml) 2070 01772 .4 140.0 + 80.1 *

Anti-dsDNA IgG2a (ng/ml) 864 7+33. 7 475.2 1 138.3 * Values meaniSEM

Student' s t-test was conducted for statistical analysis. *:p<0.05 (n=8)

<EXAMPLE 7> Effect of Sophora flavescens on the Expression of Cytokines in Spleen Lymphocyte of NZB/W Fl Mice (1) Material and Method

1) Preparation of Sophora flavescens extract and NZB/W Fl mice

A Sophora flavescens extract was prepared as in (1) of Example 1 and NZB/W Fl mice as in (2) of Example 1.

2) Preparation of Spleen lymphocytes

The spleens were excised from the NZB/W Fl mice and homogenized using a sterile syringe. Following filtration through a cell strainer (BD Bioscience, San Diego, CA, USA) , the homogenized cells were incubated with 5 ml of PharM Lyse (BD Bioscience, San Diego, CA, USA) for 5 min to remove erythrocytes from splenocytes.

RPMI-1640 (BD Bioscience, San Diego, CA, USA) supplemented with 10% FBS (JRH BIOSCIENCES, Lenexa, KS, USA) , 1% penicillin/streptomycin (BD Bioscience, San Diego, CA, USA) , 1OmM HEPES (JRH BIOSCIENCES, Lenexa, KS, USA) , and 2g sodium bicarbonate (JRH BIOSCIENCES, Lenexa, KS, USA) was used as a cell culture medium.

5 ml of the medium was added to a cell suspension in a tube, which was then centrifuged at 1,000 rpm for 10 min.

The cell pellet thus formed was suspended in 1 ml of the medium and stained with trypan blue in order to make cell counts. 3) Culture of spleen lymphocyte

Spleen lymphocytes, separated as in 2), were plated at a density of 2^χlO⁶ cells/ml/well in 24-well plates. The spleen lymphocytes were seeded in plates coated with 2 μg/ml of anti-CD3e antibody (clone: 145-2C11, BD

Bioscience, San Diego, CA, USA) and then co-stimulated with

2 μg/ml of anti-CD28 (clone: 37.51, BD Bioscience, San

Diego, CA, USA) . The plates were incubated at 37⁰C for 48 hours in a 5% CO₂ incubator (Nuaire, Plymouth, MN, USA) and the supernatant was stored at -20⁰C before use in test.

4) Measurement of cytokine expression using ELISA The levels of IFN-γ and IL-4 in the culture media of

NZB/W Fl mice were measured using an OptEIA mouse IFN-γ set and an OptEIA mouse IL-4 set, both from BD Bioscience, San

Diego, CA, USA, respectively. To this end, a dilution of a capture antibody (anti-mouse IFN-γ or IL-4) in a coating buffer (0.1M carbonate, pH 9.5) was plated at an amount of

100 μl per well in 96-well plates and left overnight at 4°C. The plates thus coated with the capture antibody were washed three times with a wash buffer (PBS/Tween-20 0.05%).

200 μl of Assay Diluent (BD Bioscience, San Diego, CA, USA) was added to each well of the plates, followed by incubation at room temperature for 1 hour for blocking. Three washings with a wash buffer preceded incubation with

100 μl of a control or a sample per well at room temperature for 2 hours . Five washings with the wash buffer was followed by the addition of 100 μl of a working detector (antibody + avidin-HRP) per well and incubation at room temperature for 1 hour. Again, the plates were washed 10 times with the wash buffer before the addition of 100 μl of a substrate solution (TMB Substrate Reagent; Pharmingen, BD Bioscience, San Diego, CA, USA) per well and incubation for 30 min at room temperature in a dark condition (in the absence of light) . 30 min after the addition of 50 μl of 2N H₂SO₄ per well, absorbance was read at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA) .

(2) Result

From the spleens of the three-month-old NZB/W Fl mice which were orally administered with the 1% Sophora flavescens extract for 15 weeks, spleen lymphocytes were separated and cultured for 48 hours. The cell culture media were assayed for cytokine expression using ELISA.

The test group was decreased in IFN-γ level (10670.0+1886.0 pg/ml) , compared with the control (18790.0+3319.0 pg/ml), with statistical significance. The level of IL-4 was measured to be 1013.0±218.3 pg/ml in the test group, which was increased as compared to the control (614.5153.4 pg/ml), but this was not significant (TABLE 5, FIG. 10) . TABLE 5

Effect of Sophora flavescens on Expression of IFN-γ and IL- 4 in Spleen Lymphocyte of NZB/W Fl Mice

Values mean±SEM

Student's t-test was conducted for statistical analysis. *: p<0.05 (n=8)

<EXAMPLE 8> Effect of Sophora flavescens on Renal Function of NZB/W Fl Mice

The 3-month-old NZB/W Fl mice of (2) of Example 1 were orally administered with the 1% Sophora flavescens extract solution of (1) of Example 1 for 15 weeks and then sacrificed. Kidney tissues excised from the test group and the control were washed with saline, fixed with 10% neutralizing formalin, washed, dried and embedded in paraffin. The formalin-fixed, paraffin-embedded tissue was sliced at 4 μm and stained according to the protocol of PAS (Periodic Acid-Schiff staining system, Sigma, ouis, MO, USA) .

PAS (Periodic acid-Schiff) staining indicated that the test group underwent less glomerular injury than did the control, and no macrophage infiltration (FIG. 11) .

Industrial Applicability

As described hitherto, the Sophora flavescens extract according to the present invention is applicable to the treatment of lupus diseases thanks to its ability to suppress SLE-induced glomerular injury and macrophage infiltration.

In addition, the Sophora flavescens extract according to the present invention is found to decrease the level of anti-dsDNA total IgG as well as IFN-γ, both main factors indicating autoimmune diseases. Therefore, the Sophora flavescens extract is useful in the treatment of autoimmune diseases including rheumatoid arthritis, multiple sclerosis, immune mediated or type 1 diabetes mellitus, inflammatory bowel disease, and scleroderma.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

1. A pharmaceutical composition for the treatment of an autoimmune disease, comprising a Sophora flavescens extract .

2. The pharmaceutical composition according to claim 1, wherein the autoimmune disease is systemic lupus erythematosus .

3. The pharmaceutical composition according to claim 1, wherein the autoimmune disease is rheumatoid arthritis.

4. The pharmaceutical composition according to claim 1, wherein the autoimmune disease is multiple sclerosis.

5. The pharmaceutical composition according to claim 1, wherein the autoimmune disease is immune mediated or type 1 diabetes mellitus.

6. The pharmaceutical composition according to claim 1, wherein the autoimmune disease is inflammatory bowel disease.

7. The pharmaceutical composition according to claim 1, wherein the autoimmune disease is scleroderma.

8. A food composition comprising a Sophora flavescens extract, useful in the treatment of an autoimmune disease.

9. The food composition according to claim 8, wherein the autoimmune disease is systemic lupus erythematosus .

10. The food composition according to claim 8, wherein the autoimmune disease is rheumatoid arthritis.

11. The food composition according to claim 8, wherein the autoimmune disease is multiple sclerosis.

12. The food composition according to claim 8, wherein the autoimmune disease is immune mediated or type 1 diabetes mellitus.

13. The food composition according to claim 8, wherein the autoimmune disease is inflammatory bowel disease.

14. The food composition according to claim 8, wherein the autoimmune disease is scleroderma.

15. A food composition comprising the therapeutic agent for autoimmune disease of claim 8, useful in the treatment of systemic lupus erythematosus (SLE) .