WO2003094947A1

WO2003094947A1 - Herbal extract for prophylaxis or treatment of inflammatory diseases and process for preparation thereof

Info

Publication number: WO2003094947A1
Application number: PCT/KR2002/000862
Authority: WO
Inventors: Hyung Min Kim
Original assignee: Chemon Inc.
Priority date: 2002-05-09
Filing date: 2002-05-09
Publication date: 2003-11-20
Also published as: AU2002307677A1

Abstract

The present invention relates to herbal extract for prophylaxis or treatment of inflammatory diseases and process for preparation thereof. Particularly, the present invention relates to water extract or alcohol extract of the mixture of Catalpa ovata, Caragana sinica, Achyranthes bidentata, Acanthopanax sessiliflorus SEEM and Glycyrrhiza uralensis FISCH, and compositions comprising the same for the prophylaxis or treatment of inflammatory nerve and joint diseases consisting of degenerative nerve arthritis, rheumatoid arthritis, and arthritis caused by physical injury.

Description

HERBAL EXTRACT FOR PROPHYLAXIS OR

TREATMENT OF INFLAMMATORY DISEASES AND

PROCESS FOR PREPARATION THEREOF

FIELD OF THE INVENTION

The present invention relates to herbal extract for prophylaxis or treatment of inflammatory diseases and process for preparation thereof. Particularly, the present invention relates to water extract or alcohol extract of the mixture of Ca talpa ova ta , Caragana sinica , Achyranthes bidentata, Acanthopanax sessiliflorus SEEM and Glycyrrhiza uralensis FISCH, and compositions comprising the same for the prophylaxis or treatment of inflammatory nerve and joint diseases consisting of degenerative nerve arthritis, rheumatoid arthritis, and arthritis caused by physical injury.

BACKGROUND OF THE INVENTION

A joint consists ^'of articular cartilage, bone therearound, ligament and synovial membrane covering the joint. Joint related inflammatory diseases can be classified into chronic rheumatoid arthritis caused by self-immunity, infectious arthritis caused by bacterial infection, deforming arthritis characterized by denaturation or destruction of articular cartilage or bone caused by various reasons, and crystal arthritis characterized by deposition of soluble metabolites as crystals inside connective tissue around joint caused by degenerative change of connective tissue. Degenerative change going with the ageing starts from articular cartilage. When chondrocytes producing cartilage components become older, they lose their functions, which is that cartilage and membrane covering joint become less flexible not to protect the joint from external shock. As this condition is going further, cartilage surface become rough and inflammation develops repeatedly by various materials flowing into joint "cavity. In addition, articular cartilage repeats the process of destruction and generation. As destroyed cartilage is superior in its numbers to generated cartilage, the amount of articular cartilage absorbing shocks decreases and even be lost. Then, bones between joints meet each other, resulting in evoking serious pain.

Although degenerative arthritis, namely osteoarthritis, is the most common joint disease and is an inevitable disease accompanied by ageing, its causes are not clearly disclosed yet. It is just supposed that the generally known causes for arthritis work together and some specific reasons are considered to affect in addition to them. The long-term exposure to pathopysiological process in childhood results in the high frequency of degenerative arthritis in old age. The generally believed causes of degenerative arthritis are as follows:

1) Ageing - ageing is not the only reason for osteoarthritis, but it interrupts blood flowing through joint area and reduces the speed of bone regeneration around bone-cartilage junction sites. Ageing is also lowering the function of peripheral nerve, which is regarded to be one of the causes of osteoarthritis.

2) The primary transformation of cartilage matrix, function of chondrocyte metabolism and metabolism regulators is also a major reason for degenerative arthritis. Meanwg-le,, incomplete recovery from mechanical damage or aftereffects of inflammatory joint diseases could be another reason.

3) Obesity - obesity greatly increases a load of joint by heavy weight, and induces a change of posture and walking. When this state continues, degenerative arthritis is progressing.

However, with the development of molecular biology, different .approaches have been tried to understand inflammatory nerve and joint diseases based on immunological level such as cytokine. And major factors influencing the above diseases have been clarified one by one. Especially, the study on cytokine secreted by mast cells is drawing an attention most . Mast cells are known to be related to mainly an allergic reaction or various inflammatory reactions. In these cells, IL-8, IL-13, TNF- α , GM-CSF (granulocyte acrophage colony stimulating factor) , IFN-γ, leukemia inhibitory factor, etc are secreted or the expression of each mRNA is increased. These cytokines not only regulate the generation of IgE antibody and immunoreaction but also play an important role in the development of inflammatory reaction with hematopoietic cells, tissues and blood vessels reaction. Among the cytokines secreted from mast cells, IL-1, IL- 6 and TNF- α are very important or inducing inflammatory reaction,. ^specially, TNF- α and IL-1 are confirmed to be major factors to cause rheumatoid arthritis, which means the regulation of those cytokines are crucial for the treatment of rheumatoid arthritis. These cytokines stimulate acute reactive- protein genes to produce proteins and corpuscular factors to activate inflammatory cells. As one of major factors causing inflammation, TNF- α induces loss of nerve by damaging tissues. Over-production of IL-β contributes to the development of various diseases and especially, relates to tϊie development of various kinds of malignant tumors, proliferative diseases, autoimmune diseases and infectious diseases. The expression of TNF- α and IL-β depends on the activation of NF-KB, a transcription element. At least 5 different genes such as NFKB1 (pl05/50) , NFK B2 (pl00/52) , ReA(p65), Rel B and c-Rel belong to NF- B family. Generally, NF-KB is composed of a dimer consisted of NF Bl(p50) and Rel A(pβ5) or NFκBl(ρ50) and NFκB2(p52). NF-KB exists in cytoplasm by being combined with internal inhibitor I B. When the decomposition and phosphorylation of IκB are induced by any specific signal, it moves into a nucleus and is activated by combining with a specific DNA sequence located in a promoter of a gene such as TNF- α and IL-6.

Meanwhile, pharmaceutical compounds for the treatment of nervous arthritis are classified by the three major workin,g .mechanisms as follows; i) mitigation of inflammation, ii) retardation of disease progression and iii) diminution of uric acid concentration. Lots of pharmaceutical compounds for the treatment of nervous arthritis have been used for mitigating inflammation. Inflammation is a pathological process causing pain, edema, fever, flare and stiffness. Non-steroid antiphlogistic agents as aspirin and steroids as cortison are used to mitigate inflammation promptly. Aspirin or other anodynes and non-steroid antiphlogistic agents can relieve pain and inflammation of nervous joint, leading to rather comfortable movement of nervous joint. However, these drugs can cause side effects such as gastrointestinal trouble or stomachache and even bring serious damages to a human body by long-term administration. Thus, patients having gastrointestinal ulcer or hemorrhage should avoid these drugs. In case of steroid hormones, their side effects such as weight gaining and hypertension are bigger than their treatment effect, so that they have not been used often for the treatment of degenerative nervous arthritis. Only when the inflammation in knee joint is serious and other treatment agents do not work so well, the injection of steroids into the joint is allowed. The injection of steroids, though, has nothing to do with the treatment of the disease but simply reduce pain, which could be resulted in worse condition of the destruction and the disorder of nervous joint by excessive use of the joint owing to reduced pain. Therefore, it is required to develop a novel pharmaceutical compound for the prevention or the treatment of inflammatory nerve and joint diseases without side effects. Thus, the present inventors have studied to develop a novel pharmaceutical compound for the prevention or the treatment of inflammatory nerve and joint diseases without any side effects and have accomplished the present invention by confirming that the water extract or the alcohol extract of the mixture of Ca talpa ova ta , Caragana sinica , Achyranthes bidenta ta , Acanthopanax sessiliflorus SEEM and Glycyrrhiza uralensis FISCH has an excellent effect as a pharmaceutical compound for the prevention or the treatment of inflammatory nerve and joint diseases without former problems.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide novel herbal extracts which have an excellent efficacy on inflammatory nerve and joint diseases such as degenerative arthritis, rheumatoid arthritis, nervous arthritis caused by mechanical injury, or inflammatory backache without any side effects, and the preparation method thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effects of the wine extract of the present invention on cell viability in HMC- 1 cells ,

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

FIG. 2B is a graph showing the effects of the wine extract of the .present invention on TNF- α secretion in HMC-1 cells,

FIG. 3A is a graph showing the time course of IL-

1J3 secretion from HMC-1 cells,

FIG. 3B is a graph showing the effects of the wine extract of the present invention on IL-1J3 secretion in HMC-1 cells,

FIG. 4A is a graph showing the time course of IL- 8 secretion from HMC-1 cells,

FIG. 4B is a graph showing the effects of the wine extract of the present invention on IL-8 secretion in HMC-1 cells,

FIG. 5 is a set of electrophoresis photographs showing the effects αf the wine extract of the present invention on the expression of TNF- α , IL-ljβ and IL-8 in HMC-1 cells .

A : Expression of TNF- α mRNA 7 hour incubation after PMA stimulation,

B : Expression of IL-lβ RNA 5 hour incubation after A23187 stimulation,

C : Expression of IL-8 mRNA 5 hour incubation after A23187 stimulation.

DETAILED DESCRIPTION' OF PREFERRED EMBODIMENTS

The present invention provides a water extract or an alcohol extract of the mixture of Ca talpa ova ta , Caragana sinica, Achyranthes bidentata, Acanthopanax sessiliflorus SEEM and Glycyrrhiza uralensis FISCH for the prophylaxis or the treatment of inflammatory diseases.

The present invention also provides a wine extract obtained by fermentation after adding the mixture of steamed rice, Nuruk and dry yeast to the above extracts for the prophylaxis or the treatment of inflammatory diseases.

The present invention further provides the preparation method of the above extracts.

The present invention also provides a pharmaceutical composition comprising the above extracts as an effective ingredient for the prophylaxis or the treatment of inflammatory diseases. Hereinafter, the present invention is described in detail.

In one aspect, the present invention provides a water extract or an alcohol extract of the mixture of Catalpa ova ta , Caragana sinica , Achyranthes bidentata, Acanthopanax sessiliflorus SEEM and Glycyrrhiza uralensis FISCH for the prophylaxis or the treatment of inflammatory diseases. For the above extract, alcohol is selected from the group consisting of 10-100% methanol, ethanol, propanol and butanol. Especially, it is preferable way to extract for 5-10 hours after adding 70-99.9% ethanol. To prepare the extract of the present invention, the preferable weight ratio of Ca talpa ova ta , Caragana sinica , Achyranthes bidenta ta, Acanthopanax sessiliflorus SEEM and Glycyrrhiza uralensis FISCH is 0.5-1 : 0.5-1 : 0.5-1 : 0.1-1 : 0.05-0.1.

The composition ratio of the above-mentioned herbs is resulted fr^*om "repeated experiments. If the ratio of any ingredient goes lower than the minimum level, the efficacy of the composition decreases and if goes higher than the maximum level, it drops the efficacy of other ingredients, resulting in weakness of synergism and interaction of those ingredients. Ca talpa ova ta, the first effective ingredient of the present invention, belongs to Bignoniaceae . Ca talpa ova ta is an about 6 m tall tree having egg-like wide brown leaves and light gray bark. Light yellow flower blooms at the end of branch in July. It is originated from China and planted in a garden. It was written in the Botanical list that Ca talpa ova ta is the best of 100 kinds of trees, so that it was called the king of trees. It was also called "thunder divine tree" since it had never been hit by a stroke of lightning, which people regarded it as a divine protection. The tree was mainly used for the construction of a royal^' palace or a temple. It has been traditionally used for the treatment of nephritis, peritonitis, uremia, edema, etc and used as a raw material of a diuretic agent as well. Recently, it is further applied for the treatment of liver cancer, liver cirrhosis, leukemia, etc.

The second effective ingredient of the present invention, Caragana sinica belongs to pea family

{ Leguminosae) and the root of Caragana sinica has been traditionally used as a medicine for helping weak constitution or relieving edema with its diuretic and vital energy-invigorating effect. It has been also known to control the symptoms of wind-dampness and arthralgia owing to its effect of promoting blood flow and alleviating pain.

The third effective ingredient of the present invention, Achyranthes bidenta ta is the root of Achyranthes japonica that belongs to Amaranthaceae . It has been widely used for the treatment of amenorrhea due to blood stagnation, menstrual abdominal pain and menstrual irregularities with its effect of promoting blood circulation to remove blood stasis, and for relieving pain in the lumber and knee joint with effect of easing joint movement.

The forth-effective ingredient of the present invention, Acanthopanax sessiliflorum SEEM is bark of Acanthopanax sessiliflorum that belongs to Aralia ceae . It has been reported to be very effective in treating the symptoms of wirid-d&mpness, arthralgia, muscular contracture, etc owing to its effects of eliminating wind-dampness, strengthen liver and kidney, and forcefulness of muscle and bones.

The fifth effective ingredient of the present invention, Glycyrrhiza uralensis FISCH is root and stem of Glycyrrhiza uralensis, a kind of perennial herb. It has been known to have the effects of invigorating the spleen and augmenting the Gi, removing heat from the blood and toxic substances, moistening the lung to arrest cough, etc. It especially harmonizes various drugs and alleviates the severe characteristics of the drugs

The present invention also provides a wine extract obtained by fermentation after adding the mixture of steamed rice, Nuruk and dry yeast to the water extract or the alcohol extract.

In the preferred embodiments of the present invention, the preferable weight ratio of Ca talpa ova ta , Caragana sinica, Achyranthes bidentata , Acanthopanax sessiliflorus SEEM and Glycyrrhiza uralensis FISCH is 0.5-1 : 0.5-1 : 0.5-1 : 0.5-1 : 0.05-0.1 and the preferable weight ratio of rice, Nuruk and yeast is 1- 4 : 0.2-1 : 0.02-0.1. It is recommended to produce the compositions of the present invention as a liquid formulation.

The present inve tion further provides the preparation method of the above water or alcohol extract and a wine extract. The preparation method of the water or alcohol extract of the present invention, comprising steps of:

1) boiling the mixture of Ca talpa ova ta , Caragana sinica, Achyranthes bidentata, Acanthopanax sessiliflorus SEEM and Glycyrrhiza uralensis FISCH after adding water or alcohol therein; and

2) concentrating liquid part isolated from solid part of the above mixture.

At this time, alcohol is selected from the group consisting of 10-100% methanol, ethanol, propanol and butanol. Especially, it is preferable way to extract for 5-10 hours after adding 70-99.9% ethanol.

The preparation method of the wine extract of the present invention, comprising steps of:

1) fermenting the above water extract or alcohol extract after adding the mixture of rice, Nuruk and dry yeast therein; and

2) concentrating liquid part isolated from solid part of the above mixture.

At this time, it is no problem to use generally consumed rice but preferable to use steamed rice.

The present invention also provides a pharmaceutical composition containing the above water extract, alcohol extract or the wine extract as effective ingredients for the prophylaxis or the treatment of inflammatory diseases.

The pharmaceutical composition of the present invention can be effectively used for inflammatory diseases such as degenerative nerve arthritis, rheumatoid arthritis, arthritis caused by physical injury, and inflammatory lumbago, but not be limited in use for those group. The composition of the present invention restrains local and general inflammatory reaction by regulating the generation of cytokines and chemical mediators in immune cells (see Experimental Example 1 - 3) .

In order to prepare the pharmaceutical composition of the present invention, the extract obtained by decocting or extracting the above medicinal herbs with solvents according to the physical/chemical characteristics of their acting compounds or the powder obtained by concentrating and drying the extract were mixed with pharmaceutically allowed carriers, which was then produced as tablets, granules, or liquid medicines following the general manufacturing process. It is preferable to produce the pharmaceutical composition of the present invention as a liquid medicine, which shows better efficacy. But if necessary, the composition could be produced in the forms of tablets, granules, sprays, pills, coated tablets or capsules.

To prepare the composition of the present invention, 12,000 ml of water was added to the mixture of 0.5-1 kg of Catalpa ova ta, 0.5-1 kg of Caragana sinica , 0.5-1 kg of Achyran thes bidenta ta, 0.1-1 kg of Acanthopanax sessiliflorus SEEM and 0.05-01 kg of Glycyrrhiza uralensis FISCH, which was decocted for about two and half hours and then concentrated up to 8,000 mi, resulting in obtaining the extract therefrom. The extract was regulated to meet the volume of 10,000 mi by following the steps such as 1) the extract was added to the mixture of steamed rice produced from 1-4 kg of rice, 0.2-1 kg of Nuruk and 0.02-0.1 kg of dry yeast, 2) fermented thereof at 20-27^°C for 3-10 days, 3) filtered thereof to prepare wine extract, and 4) added distilled water until its volume reached 10,000 mi . The dosage of the composition of the present invention is generally 1.5-2.5 mi/kg and it is preferred to be administered three times a day. However, it may be necessary to deviate from the dosages mentioned, and in particular to do so as a function of the nature and body weight of the object to be treated, the nature, and severity of the disease, the nature of the formulation and of the administration of the medicament, and the period or interval within which administration takes place.

For instance, a 50 kg weighing adult is required to take it 90~150 mi each time, three times a day. The above prepared composition has about 20~25 day worth amount, but the dosage can be increased or decreased by the weight, age, gender, digestive condition of a patient, and severity of the disease. The above dosage of a liquid composition can be a good reference for other formulations, and they can be administered by orally or parenterally. In general, from 2-3 days after administration, symptoms begin to be relieved, though there is an individual variation. When the composition is coming into effect, at least one month long administration is required from then on.

The pharmaceutical composition of the present invention for the prophylaxis or the treatment of inflammatory diseases can contain generally used fillers, extenders, binders, wetting agents, disintegrating agents, diluents such as surfactant, or excipients additionally. The composition of the present invention can be formulated as tablets, capsules, pills, granules, suspensions, emulsions, and other liquid formulations.

Particularly, the composition of the present invention can be prepared as formulations for oral administration such as tablets, troches, lozenges, suspensions, dusting powders, granules, emulsions, hard or soft capsules, syrups, and elixirs. Tablets, coated tablets, capsules, pills, and granules can contain the active compound or compounds in addition to the customary excipients, such as fillers and extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; binders, for example, carboxymethylcellulose, alginates, gelatine, and polyvinylpyrrolidone; humectants, for example, glycerol; disintegrating agents, for example, agar-agar, calcium carbonate, and sodium carbonate; solution retarders, for example, paraffin; absorption accelerators, for example, quaternary ammonium compounds; wetting agents, for example, ethyl alcohol and glycerol monostearate; adsorbents, for example, kaolin and bentonite; and lubricants, for example, talc, calcium stearate, magnesium stearate, and solid polyethylene glycols.

In addition, the pharmaceutical composition of the present invention can be administered by parenterally. This way includes subcutaneous injection, intravenous injection, intramuscular injection, intracisternal injection, intravaginal injection, or intraperitoneal injection. In order to prepare the composition as a formulation for parenteral administration, the extract is required to be mixed with stabilizers or buffers in water to be made as solutions or suspensions, which are finally prepared by the dosage units of ample or vial. The pharmaceutical composition of the present invention was administered orally to rats with the dosage of 25 mi/kg for the toxicity test. As a result, the composition has been confirmed not to have any toxicity or any side effect on liver or any other organs .

EXAMPLES

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Preparation of water extract

Twenty-five grams of Ca talpa ova ta , 25 g of Caragana sinica, 25 g of Achyranthes bidenta ta, 22.5 g of Acanthopanax ses.sil j.florus SEEM and 2.5 g of Glycyrrhiza uralensis FISCH were extracted with 1,000 mi of distilled water at 100^°C for about 2 hours. About 200 mi of water extract was obtained.

Example 2 : Preparation of wine extract

Seven hundred fifty grams of Ca talpa ova ta , 0.75 kg of Caragana sinica , 0.75 kg of Achyranthes bidentata , 0.675 kg of Acanthopanax sessiliflorus SEEM and 0.075 kg of Glycyrrhiza uralensis FISCH were extracted with 12,000 mi of distilled water at 100^°C for about 2.5 hours. About 8,000 mi of water extract was obtained. Besides, 4 kg of steamed rice, 1 kg of Nuruk (Korean traditional malted wheat) and 0.05 kg of dry yeast were mixed homogeneously. The mixture of Nuruk, yeast and steamed rice was immersed by the water extract, and then fermented at 10-25^°C. After 3 days fermentation, the fermented solution was filtered to separate liquid part and solid part. After warming the liquid part at

55°C-60°C and adding distilled water, 10,000 ml of wine extract was prepared.

Example 3: Preparation of wine extract 2

Seven hundred fifty grams of Catalpa ovata , 0.75 kg of Caragana sinica , 0.75 kg of Achyran thes bidentata ,

0.675 kg of Acanthopanax sessiliflorus SEEM, 0.075 kg of

Glycyrrhiza uralensis FISCH, 4 kg of steamed rice, 1 kg of Nuruk and 0.05 kg of dry yeast were mixed homogeneously. After adding 18,000 mi of distilled water to the above mixture, the mixture was fermented at 10-25^°C. After 5 days fermentation, the fermented solution was filtered to separate liquid part and solid part. After warming the liquid part at 55^°C-60^°C and adding distilled water, 10,000 mi of wine extract was prepared.

Example 4: Preparation of alcohol extract

Seventy-five grams of Catalpa ova ta , 75 g of Caragana sinica, 75 g of Achyranthes biden ta ta , 67.5 g of Acanthopanax sessiliflorus SEEM and 7.5 g of Glycyrrhiza uralensis FISCH were extracted with 2,000 mi of ethanol for about 6 hours to obtain alcohol extract .

Example 5: Preparation of other formulations

The present inventors have prepared pills, granules, aerosols, t blets and capsules by evaporative drying, concentrating or mixing the extracts obtained in the above Example 1-4 with complementary agents according to the known methods.

Experimental Example 1: Acute toxicity and side effect

test

The following experiments were performed to see if the herbal extracts pf the present invention have acute toxicity in rats.

6-week old SPF SD line rats were used in the tests for acute toxicity. Herbal extracts obtained in the examples of 1~4 were orally administered to 30 rats for 90 days at the dosages of 2.5, 12.5 and 25 mi/ kg. Death, clinical symptoms, and weight change in rats were observed, hematological tests and biochemical tests of blood performed, and any abnormal signs in the gastrointestinal organs of chest and abdomen checked with eyes during autqpsy The results showed that the herbal extracts did not cause any specific clinical symptoms, weight change, or death in rats. No change was observed in hematological tests, biochemical tests of blood, and autopsy. The herbal extracts used in this experiment are evaluated to be safe substances since they do not cause any toxic change in rats up to the level of 25 mi/kg and their estimated LD₅₀ values are much greater than 25 mi/kg in rats.

Experimental Example 2: Secreted cytokine levels in

human leukaemic mast cell line

Many kinds of studies about inflammatory neuroarticular disease have been performed because of its various cause. Among them, determination of secreted cytokine levels in human leukaemic mast cells (HMC) can be a good parameter for the study of the disease. Among the cytokines secreted from HMCs, TNF- α , IL-1 and IL-6 are the kinds of inflammatory cytokine. Therefore, if a medicinal drug suppresses the expression of these cytokines, it can be concluded that the drug has anti-inflammatory effect. Thus, the present inventors treated HMC-1 cell line (obtained from National Institute of Health, USA) with the herbal extracts of the present invention, and then observed the secreted levels of TNF- α , IL-1 and IL-6 in the HMC-1 cells.

Particularly, to determine whether the water extract prepared in Example 1, the wine extract prepared in Example 2 and 3, and the alcohol extract prepared in Example 4 can regulate expression of TNF- α and interleukins produced by IgE-Ag stimulation, the present inventors treated HMC-1 cells with the above herbal extracts. Secreted TNF- α , IL-1 and IL-6 levels in culture supernatant of HMC-1 were measured by a sandwich enzyme-linked immunosorbent assay (ELISA) .

Monoclonal antibodies to cytokines, purified anti-murine TNF- α , IL-1 and IL-6 were diluted with PBS(pH 7.4) and fixed on a 96-well plate. The plate was washed with PBS containing 0.05% Tween. Blocking reaction was performed for 1 hour by adding PBS containing 1% BSA, 5% sucrose and 0.05% NaN₃. After washing several times, culture supernatant obtained by centrifugation and standard cytokines were added in the plate, and the plate was cultured at 37 ^°C for 2 hours. After washing, rabbit IgG-conjugated alkali phosphatase and avidin peroxidase were added into the wells of the plate. The plate was cultured again at 37 ^°C for 2 hours. The wells of the plate were washed again. After adding PNPP and ABTS substrate into the wells of the plate, absorption of avidin horseradish peroxidase color reaction was measured with ELISA reader at 405 nm. As a result, the wine extract of the present invention prepared in Example 2 almost 100% blocked the secretion of TNF- α and IL-6, and blocked the IL-1 secretion by 44% inhibition at the concentration of 0.1 mg/mi (Table 1) .

<Table 1> Inhibition effects of the wine extract (0.1 mg/mi) of the present invention on the production of TNF- α , IL-6 and IL-1 in human HMC-1 cells

The herbal extracts prepared in Examples 1, 3 and 4 were also inhibited the secretion of cytokines in HMC cells. However, the inhibition level was lower than that of the wine extract prepared in Example 2. These results indicate that the water extract prepared in Example 1, the wine extract prepared in Examples 2 and 3, and the alcohol extract prepared in Example 4 can regulate the secretion of TNF- α , IL-6 and IL-1.

From the above results, it was confirmed that the herbal extracts of the present invention can be effectively used for the prophylaxis or the treatment of inflammatory nerve and joint diseases by regulating the three cytokines related with inflammatory reaction by the inhibitory mechanism of the expression of TNF- α , IL-6 and IL-1.

Experimental Example 3: Expression of cytokines in PBMC

isolated from human peripheral blood

The present inventors examined the effects of the wine extract of the present invention prepared in Example 2 on the expression of cytokines in human leukaemic mast cells (HMC-1) and peripheral blood mononuclear cells (PBMC) isolated from human peripheral blood.

<3-l> Patients and isolation of PBMC

Peripheral blood was obtained from 3 patients (2 women and 1 man) with active stage of rheumatic arthritis. All patients with rheumatic arthritis (mean age 61.0+7.5 years; mean disease duration 7.7+4.5 years) met the American Rheumatism Association criteria for disease (Arnett, F. C, et al., J. Korean Soc . Food. Nutr. , 1996, 25, 170-179). To investigate the effects of the wine extract of the present invention on specificity for rheumatic arthritis, the present inventors obtained peripheral bloods from age-matched 3 healthy and 3 disease (non-rheumatic arthritis) controls. Disease control group consisted of three patients who are not related to rheumatic arthritis, and they had liver cirrhosis, cerebral infarction, and post cerebral infraction, respectively. Peripheral blood was collected into EDTA-containing tubes. To minimize the in vi tro effect on the activation status of the cells, mononuclear cells were immediately purified by rapid Ficoll-Hypaque (Histopaque-1077 ) centrifugation at room temperature. PBMCs were harvested from the interface layer, washed twice with PBS and once more with RPMI 1640 medium. PBMCs were stabilized in RPMI 1640 medium for 2 hours at 37 ^°C in 5% C0₂ incubator. Stabilized PBMCs (6X10⁵ cells/ml) were seeded in culture plates.

<3-2> Cell culture and stimulation

Human leukaemic mast cell line (HMC-1) was grown in IMDM (Iscove's modified Dulbecco's medium, Gibco BRL) supplemented with 100 units/mi of penicillin, 100 βg/ml of streptomycin, 10^"5 M of monothioglycerol and 10% heat-inactivated fetal bovine serum(FBS) at 37°C in 5% C0₂. HMC-1 cells (I 10⁵ cells/ml) were treated with the wine extract (1-100 βg/ml) of the present invention prepared in Example 2 for 30 minutes prior to stimulation with 20 nM of phorbol 12-myristate 13- acetate (PMA, Sigma) or 1 μM of A23187 and incubated at 37 ^°C for additional time. Human PBMCs were grown in RPMI 1640 medium supplemented with 100 units/ml of penicillin, 100 βg/ml of streptomycin and 10% heat- inactivated FBS. PBMCs (6X10⁵ cells/mi-) were pretreated with the wine extract (0.1-10 βg/ml) of the present invention and then stimulated with 25 βg/ml of phytohaemagglutinin (PHA_f Sigma) and incubated for 24 hours .

<3-3> MTT assay

To investigate the viability of cells, the present inventors performed MTT colorimetric assay (Kim, M. S., et al., Cancer Lett . , 2001, 171, 75-89). Briefly, HMC-1 cells (5X10⁴ cells/ml) were incubated for 8 hours after stimulation with PMA or A23187 in the absence or presence of the wine extract of the present invention. After addition of MTT solution, the cells were incubated for 4 hours. The crystallized MTT was dissolved and measured the absorbance at 540 nm.

Results were expressed as the mean+SEM of the indicated number of experiments. The significance of the differences between the PMA- or A23187-treated groups and the control group, and between the PMA- or A23187-treated groups and the wine extract of the present invention-treated groups were determined by the Mann-Whitney U test.* Differences among the rheumatic arthritis group, disease control, and healthy control were compared by the analysis of variance (ANOVA) , with post-hoc test of the means according to Tukey' s method. For all tests, P values less than 0.05 were considered significant .

As a result, the wine extract of the present invention had no toxic effects on HMC-1 cells viability. FIG.l shows the viability of cells 8 hour incubation after stimulation with 20 nM of PMA or 1 μM of A23187 in the absence or presence of the wine extract of the present invention. In the cells treated with PMA or A23187, cell viability slightly decreased to 87.5+7.0% or 91.4+1.6% compared with control value, 100+4.0%. However, the wine extract of the present invention didn't affect cell viability in each condition and had no toxicity on HMC-1 cells. <3-4> Measurement of effects on cytokine secretion

Secreted cytokine levels in culture supernatant of HMC-1 or PBMC were measured by a sandwich enzyme- linked immunosorbent assay (ELISA) according to manufacture's protocol (for TNF- α and IL-lJβ assay, R&D Systems; IL-8 assay, PharMingen) . Absorption of the avidin-horseradish peroxidase color reaction was measured at 405 nm and compared with serial dilutions of human recombinants as a standard.

As a result, the wine extract of the present invention inhibited the secretion of TNF- α , IL-lβ and IL-8 from HMC-1 cells. HMC-1 cells can be activated by non-physiological agents such as PMA or A23187 and release the various cytokines (Queralt, M. , et al., Inflamm . Res . , 2000, 49, 355-360; Kruger-Krasagakes, S., Immunol . , 1999, 98, 253-257). In preliminary experiments, the present inventors observed the optimal time condition for secretion of proinflammatory cytokines, TNF- α , IL-lβ and IL-8, by PMA or A23187. TNF- α secretion reached a peak 8 h incubation after 20 nM PMA stimulation (FIG. 2A) . Over ten-fold increase of TNF- α was obtained in the supernatant after 8 h stimulation compared with no stimulation. The present inventors examined the inhibitory effects of the wine extract of the present invention on TNF- α secretion in PMA-induced HMC-1 cells (FIG. 2B) . The wine extract of the present invention inhibited TNF- α secretion in a dose-dependent manner, and the pretreatment of the wine extract (100 βg/ml) almost fully blocked the TNF- α secretion (87% inhibition). FIG. 3A shows IL-lβ released after HMC-1 cell stimulation with 1 μM of A23187. The amount of IL-lβ released into the supernatants after 6 h stimulation was shown almost three-fold increase compared with no stimulation. The influence of the wine extract of the present invention on the IL-lβ secretion from HMC-1 cells was shown in FIG. 3B. The wine extfact of the present invention inhibited IL-lβ secretion in a dose-independent manner, and the treatment of 10 βg/ml wine extract only presented statistical significance. As shown in FIG. 4A, the present inventors determined on the time point that corresponded to the burst of IL-8 secretion after stimulation with 1 μM of A23187. IL-8 secretion reached a maximum induction at 6 h incubation after stimulation and about fifteen-fold increase was shown in the supernatant compa'red with no stimulation. The effects of the wine extract of the present invention on IL-8 secretion were evaluated in FIG. 4B. The wine extract of the present invention inhibited IL-8 secretion in a dose-dependent manner and the pretreatment of 100 βg/\wl wine extract blocked by 84% inhibition.

<3-5> Isolation of RNA and RT-PCR analysis

The present inventors extracted mRNA from the harvested cells using Micro mRNA purification kit (Amersha Pharmacia Biothch) and 100 ng of mRNA was converted to cDNA by reverse transcriptase at 37 ^°C for

1 h 30 min using First-strand cDNA synthesis kit (Amersham Pharmacia Biotech) . PCR amplification was consisted of 35 cycles (94°C, 45 sec; 60°C, 45 sec; 72^°C, 1.5 min) with the commercial oligonucleotides primer sets for human TNF- α , IL-8 and β-actin. The primer sequences for IL-lβ were., taken from the publication of Yamamura et al. (Yamamura, M. , et al., Science, 1991, 254, 277-279), and the samples were amplified by 35 cycles (94^°C, 1 min; 65^°C, 1 min; 72^°C, 1 min). These primer sets yield PCR products of 355, 248, 200 and 661 bp for TNF- α , IL-lβ, IL-8 and β-actin, respectively. Final PCR products were electrophoresed on 2% agarose gels .

As a result, he _Awine extract of the present invention inhibited the expression of TNF- α and IL-lβ not IL-8 in HMC-1 cells. In order to determine whether the wine extract of the present invention can regulate cytokine mRNA expression, the mRNA was isolated from

HMC-1 cells and reverse-transcribed into cDNA. The cDNA was amplified by PCR with primers specific for

TNF- α , IL-lβ and IL-8, and the final PCR products were correspondingly yielded 355 bp, 248 bp, and 200 bp bands on 2% agarose gels (FIG. 5) . PCR using housekeeping gene human β-actin (661 bp) was carried out for confirming equivalency of cDNA preparation.

After incubation in PMA for 7 h, TNF- α expression was increased compared with the absence of PMA, and the wine extract of the present invention suppressed the

TNF- α expression in a concentration-dependent manner

(FIG. 5A) . The higher expression of IL-lβ was induced in A23187-stimulated cells, and the preincubation of 10 βg/ml of the wine extract of the present invention successfully blocked the IL-lβ expression in agreement with data on ELISA (FIG. 5B) . After incubation in A23187 for 5 h, IL-8 expression was increased compared with the absence of A23187. However, the wine extract of the present invention failed to suppress the IL-8 expression in A23187-stimulated cells (FIG. 5C) .

<3-6> Modulatory effects on cytokine secretion compared

between rheumatic arthritis and control subjects

To investigate the effects of the wine extract of the present invention on specificity for active rheumatic arthritis patients, the present inventors compared cytokine production by PBMC from age-matched healthy and unhealthy (non-rheumatic arthritis) controls.

As shown in Table 2, TNF- α and IL-lβ production was increased by PHA stimulation in PBMC (Mao, T. K. , et al., Life Science, 2000, 66, 1377-1386; Mayringer, I., et al., J. Immunol . Methods, 2000, 235, 33-40). TNF- α production was enhanced by 4.5 fold increase for active rheumatic arthritis group, by 3.1 fold for unhealthy control group, and by 5.9 fold for healthy control group compared with unstimulated PBMC (p<0.05). Although the pretreatment of the wine extract of the present invention (10 βg/ml) slightly inhibited TNF- α production in healthy control group (p=0.158), the wine extract of the present invention significantly inhibited TNF- α production from active rheumatic arthritis patient group in a dose-dependent manner. Treatment with PHA obviously induced production of IL-1 β by PBMC by 3.9 fold increase for active rheumatic arthritis group, by 3.7 fold increase for unhealthy control group, and by 12.7 fold increase for healthy control group (0<0.05). Treatment of the wine extract of the present invention for 30 min prior to stimulation with PHA blocked in a dose-dependent manner the production of IL-lβ by PBMC from active rheumatic arthritis group, not from control group.

Effects on cytokine production in PBMC

* : p<0.05 versus no PHA stimulation.

** : P<0.05 versus no wine extract treatment.

*** : p<0.001 versus no wine extract treatment

INDUSTRIAL APPLICABILITY

As shown above, the composition of the present invention can be effectively used for the prophylaxis or the treatment of inflammatory nerve and joint diseases such as degenerative nerve arthritis, rheumatoid arthritis, arthritis caused by physical injury, and inflammatory lumbago. Especially, the composition of the present invention has an excellent effect on the treatment of degenerative nerve arthritis often developed in or after middle age as well as relieving symptoms.

Those skilled in the art will appreciate that the concepts and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims .

Claims

What is claimed is :

1. A water extract or an alcohol extract of the mixture of Ca talpa ovata , Caragana sinica, Achyranthes bidenta ta, Acanthopanax sessiliflorus

SEEM and Glycyrrhiza uralensis FISCH for prophylaxis or treatment of inflammatory diseases

2. The water extract or alcohol extract as set forth in claim 1, wherein the alcohol is selected from the group consisting_, of 10-100% methanol, ethanol, propanol and butanol .

3. The water extract or alcohol extract as set forth in claim 2, wherein the alcohol is 70-99.9% ethanol .

. The water extract or alcohol extract as set forth in any one of claim 1-3, wherein the weight ratio of the mixture of Ca talpa ova ta, Caragana sinica , Achyranthes bidenta ta, Acan thopanax sessiliflorus SEEM and Glycyrrhiza uralensis FISCH is 0.5-1 : 0.5-1 : 0.5-1 : 0.1-1 : 0.05-0.1.

5. A wine extract for prophylaxis or treatment of inflammatory diseases prepared by fermenting the water extract or the alcohol extract of claim 1 with the mixture of rice, Nuruk and dry yeast.

6. The wine extract composition as set forth in claim 5, wherein the weight ratio of the mixture of rice,

Nuruk and dry yeast is 1-4 : 0.2-1 : 0.02-0.1.

7. A preparation method of the extract of claim 1, comprising steps of: (1) boiling the mixture of Ca talpa ova ta ,

Caragana sinica , Achyranthes bidenta ta , Acan thopanax sessiliflorus SEEM and

Glycyrrhiza uralensis FISCH after adding water or alcohol therein; and (2) concentrating liquid part isolated from solid part of the above mixture.

8. The preparation method as set forth in claim 7, wherein the weight ratio of the mixture of Catalpa ova ta, Caragana sinica, Achyranthes bidenta ta, Acanthopanax sessiliflorus SEEM and Glycyrrhiza uralensis FISCH is 0.5-1 : 0.5-1 : 0.5-1 : 0.05- 0.1.

9. A preparation method of the wine extract composition of claim 5, comprising steps of: (1) fermenting the above water extract or alcohol extract after adding the mixture of rice, Nuruk and dry yeast therein; and

(2) concentrating liquid part isolated from solid part of the above mixture.

10. The preparation method as set forth in claim 9, wherein the weight ratio of the mixture of rice, Nuruk and dry yeast is 1-4 : 0.2-1 : 0.02-0.1.

11. A composition for prophylaxis or treatment of inflammatory diseases comprising the water extract or alcohol extract of claim 1, or the wine extract composition of claim 5.

12. The composition as set forth in claim 11, wherein the inflammatory diseases are inflammatory nerve and joint diseases.

13. The composition as set forth in claim 12, wherein the inflammatory nerve and joint disease is selected from the group consisting of degenerative nerve arthritis, rheumatoid arthritis, and arthritis caused _sby physical injury.