WO2016153262A2

WO2016153262A2 - Pharmaceutical composition comprising the extract of platycodon grandiflorum enhanced effective saponin contents for treatment of rheumatoid arthritis

Info

Publication number: WO2016153262A2
Application number: PCT/KR2016/002906
Authority: WO
Inventors: Dong Seoung RYU; Sang Wook Lee; Wee Yong SOHN
Original assignee: B&C Biopharm Co., Ltd.
Priority date: 2015-03-26
Filing date: 2016-03-23
Publication date: 2016-09-29
Also published as: WO2016153262A3

Abstract

The present invention relates to a pharmaceutical composition comprising the Platycodon grandiflorum extract containing the enhanced effective saponin content as an active ingredient for the treatment of rheumatoid arthritis. The Platycodon grandiflorum extract of the present invention displays a significant therapeutic effect on rheumatoid arthritis. In particular, when the extract was administered as being coated specifically with the enteric coating agent that can be released in the intestines, in order for the extract to show the maximum effect with the minimum dose with minimum damage and side effects in the stomach by not being released in the stomach but being released and absorbed in the intestines, a significant therapeutic effect on rheumatoid arthritis was observed. Therefore, the Platycodon grandiflorum extract of the present invention can be effectively used for the treatment of rheumatoid arthritis.

Description

[DESCRIPTION]

[invention Title]

PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACT OF PLATYCODON GRANDIFLORUM ENHANCED EFFECTIVE SAPONIN CONTENTS FOR TREATMENT OF RHEUMATOID ARTHRITIS

[Technical Field]

The present invention relates to a pharmaceutical composition comprising the Platycodon grandiflorum extract having the enhanced content of Platycodin, the effective saponin, as an active ingredient for the treatment of rheumatoid arthritis and a health functional food comprising the same.

[Background Art]

Rheumatoid arthritis is a chronic inflammatory disease characterized by polyarthritis. In the early stage, inflammation is only observed in the synovial membrane covering the joint, but as it progresses the inflammation spreads to the cartilage and the bone to cause destruction and deformity of the joint. In addition to the damage in the joint, this disease can cause systemic body symptoms such as anemia, xerosis, subcutaneous nodule, pulmonary fibrosis, vasculitis, and skin ulcer .

The cause of the rheumatoid arthritis has not been disclosed, yet the autoimmunity is believed to be the major mechanism involved in the development of the disease. Autoimmunity is to attack the self instead of a foreign material invaded because of the malfunction of the immune system responsible for the defense of the self from the foreign attack. In general, genetic factors and bacteria or virus infection are considered as the causes of rheumatoid arthritis. It is also known that the disease seems to be developed easily under the physical or mental stress. Rheumatoid arthritis is an intractable autoimmune disease accompanying swelling, inflammation, spasticity, and pain in the joint and displaying systemic polyarthritis symptoms. Again, rheumatoid arthritis is a systemic disease, wherein, based on the defect in the self/non-self recognition, the body recognizes the self as non- self and accordingly attacks its own tissues and induces abnormal immune response, resulting in the inflammation in the connective tissue.

Degenerative arthritis, so called osteoarthritis, is caused by aging of the chondrocytes forming the joint. Precisely, as aging progresses, the synthesis of the joint matrix materials such as type II collagen and proteoglycan is suppressed but at the same time the synthesis of the inflammatory cytokines such as interleutik- 1 β and tumor necrosis factor-a is increased, so that the synthesis and activity of matrix metalloproteinase (MMP) that is involved in the degradation of the joint matrix are increased in the joint cells, resulting in the destruction of the joint tissue to cause degenerative arthritis.

Arthritis worsens when the degradation of the joint matrix is accelerated by the synthesis of MMP increased by the generation of nitrogen monoxide mediated by the inflammatory cytokine and the auto-proliferative cytokine synthesis by the generated nitrogen monoxide vice versa. The inflammatory cytokine also increases the generation of prostaglandin E2 , the lipid metabolite, to keep causing inflammatory response.

Drugs have been used for the treatment of chromic rheumatoid arthritis are exemplified by the anti- inflammatory steroids (ex. prednisolon) or non-steroid anti-inflammatory agents (ex. indometacin, aspirin, etc.), immunosuppressants (ex. cyclosporine A, tacrolimus (FK506) , methotrexate, cyclophosphamide, azathioprine , etc.), and disease modifying anti-rheumatic drugs (ex. gold salt) . The anti -inflammatory agents can regulate the inflammation and relieve pain and swelling but cannot suppress the progress of the disease. Those drugs, in fact, can only relieve the symptoms and make the progress slow but hardly treat the disease. Besides, there is a risk of side effects according to the long term administration. Therefore, any of those drugs is not satisfactory.

Drugs used for the treatment of arthritis are divided by the major mechanism of reducing inflammation, delaying the progress of the disease, and reducing the uric acid concentration. Most of the drugs for the treatment of neural arthritis can reduce inflammation. Inflammation is a pathogenic progress that causes pain, swelling, heat feeling, seizure, and spasticity. Drugs that can fast relieve inflammation are exemplified by the non-steroidal anti -inflammatory agents such as aspirin and the steroidal anti -inflammatory agents such as cortisone. '

The non-steroidal anti -inflammatory agents can reduce pain, make the neural joint smooth, and alleviate the inflammation, but might cause gastrointestinal disturbance and stomachache. Therefore, it is banned from using for the patients with active peptic ulcer or hemorrhagic disease in the digestive system. The steroidal anti- inflammatory agents display comparative more serious side effects such as weight increase or high blood pressure than its pharmaceutical effect, so that it is not often used for the treatment of degenerative neural arthritis. In particular, the steroidal anti -inflammatory agents have nothing to do with the treatment of the disease and , simply relieve pain temporarily, which might cause over-use of the joint so that the neural joint destruction and troubles might worsen. Therefore, it requires attention to use the steroidal anti-inflammatory agents .

The treatment method that have been used for arthritis so far has only limited the treatment effect but carries side effects, so that it is not appropriate for the long term treatment. Thus, it is required to develop a novel treatment method that can overcome the problem of the conventional method.

Platycodi Radix is the root of the only perennial plant Platycodon grandiflorum A. DC. belonging to Platycodon, Campanulaceae , which is distributed or cultivated in the East Asia. The medicinal herb name of it is Gilgyoung. The ingredients of Platycodi Radix are carbohydrate (sugar, more than 90%) , protein (2.4%), lipid (0.1%), and ash (1.5%). In addition, triterpenoid saponin (32 kinds of saponins have been identified such as Platycodin A, C, D, D2 , polygalacin D, D2 , etc.) is included therein at the concentration of approximately 2%. Saponin is drawing our' attention as a pharmaceutically active ingredient of Platycodi Radix. As minor ingredients in .

5 addition to the above, steroid compounds such as a-spinasterol , A7-stigmasterol, and a-spinasteryl- β-D-glucoside are included at the concentration of 0.03%. Carbohydrate therein is composed of mostly disaccharides such as glucose, fructose, sugar, and kestos or trisaccharides and some polysaccharides such as inulin and Platycodinin (corresponding to 10 fructose molecules) .

The pharmacological effects of Platycodi Radix known so far are neuroprotective effect [Yoo Ki-Yeon et al . , Neurosci. lett. 444(1), 97-101 (2008)], anti-obesity effect [Zhao, H. L. et al., J. Food. Sci. ^' 73(8), H195-H200, (2008)], hepatoprotective effect [Lee, K.J.et al . , Toxicol. Lett. 147, 271-282, (2004)], immunomodulatory activity [Ann, K.S. et al . , Life Sci. 76, 2315-2328, (2005)], and cytotoxicity [Lee, Kyung Jin et al., Food Chem. Toxicol. 46(5), 1778-1785 (2008); Zhang, Lin et al . , Molecules 12(4), 832-841, (2007)] .

To overcome the problems of the conventional commercialized drugs and the safety problem of them that have been recently a serious issue, the present inventors tried to develop a novel therapeutic agent for rheumatoid arthritis originated from safer natural materials. As a result, the inventors confirmed that the Platycodon grandiflorum extract had a significant treatment effect on rheumatoid arthritis, and particularly the inventors confirmed a significant rheumatoid arthritis treating effect from the administration of the extract with enteric coating designed to stay in the stomach not- dissolved and to be released and absorbed in the intestines in order to bring the maximum effect with the minimum dose with reducing damage and side effects in the stomach, resulting in the completion of this invention. [Disclosure]

[Technical Problem]

It is an object of the present invention to provide a pharmaceutical composition for the treatment of rheumatoid arthritis and a health functional food comprising the Platycodon grandiflorum extract as an active ingredient.

[Technical Solution]

To achieve the above object, the present invention provides a pharmaceutical composition for the treatment of rheumatoid arthritis comprising the Platycodon grandiflorum extract as an active ingredient.

The present invention also provides a health functional food for the prevention and improvement of rheumatoid arthritis comprising the Platycodon grandiflorum extract as an active ingredient .

The present invention further provides an enteric coated oral formulation comprising the Platycodon grandiflorum extract.

The present invention also provides a method for preparing the Platycodon grandiflorum extract having the enhanced effective saponin contents, which comprises the following steps:

1 ) extracting Platycodon grandiflorum by using water, Ci ~ C₂ lower alcohol, or a mixed solvent of the same;

2 ) concentrating the extract obtained in step 1 ) and separating the sugar compound from the extract by using ion exchange resin and size exclusion chromatography; and

3 ) collecting the Platycodon grandifloru extract wherein the sugar compound was separated in step 2 ) , followed by vacuum . concentration and drying to prepare the Platycodon grandiflorum powder extract.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of rheumatoid arthritis, a health functional food for the prevention and improvement of rheumatoid arthritis, and an enteric coated oral formulation, which all contain the Platycodon grandiflorum extract prepared by the method of the invention above.

[Advantageous Effect]

The Platycodon grandiflorum extract of the present invention contains Platycodin at least 1% and displays a significant therapeutic effect on rheumatoid arthritis. In particular, when the Platycodon grandiflorum extract is administered as being enteric coating in order for the extract to pass the stomach not-dissolved and to be released and absorbed in the intestines instead to bring the maximum effect with the minimum dose, it demonstrated a significant therapeutic effect on rheumatoid arthritis. Therefore, the Platycodon grandiflorum extract of the present invention can be effectively used for the treatment of rheumatoid arthritis.

[Description of Drawings]

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

Figure l is a diagram illustrating the changes in the contents of polysaccharides and crude saponin in the Platycodon grandiflorum extract.

Figure 2 is a diagram illustrating the result of pharmacokinetic experiment of Platycodin D.

Figure 3 is a diagram illustrating the saponin contents before and after the sugar separation from the Platycodon grandiflorum extract, confirmed by HPLC.

Figure 4 is a diagram illustrating the suppressive effect on the osteoclast differentiation.

Figure 5 is a schematic diagram illustrating the induction of rheumatoid arthritis in the experimental animals.

Figure 6 is a diagram illustrating the body weight changes of the rheumatoid arthritis animal model¹ by the Platycodon grandiflorum extract:

Normal group;

Control group;

the experimental group treated with the Platycodon grandiflorum extract of the present invention at the concentration of 100 mg/kg (Platycodi radix extract 100 mg/kg, PGE-100 group) ;

the experimental group treated with the Platycodon grandiflorum extract of the present invention at the concentration of 300 mg/kg (Platycodi radix extract 300 mg/kg, PGE-300 group)

the experimental group treated with the Platycodon grandiflorum extract of the present invention at the concentration of 1000 mg/kg (Platycodi radix extract 1000 mg/kg, PGE-1000 group) ; and

the positive control group treated with the crude saponin extract at the concentration of 6 mg/kg (Crude saponin extract 6 mg/kg, CS-6 group).

Figure 7 is a diagram illustrating the changes in diet and drinking water taking in the rheumatoid arthritis animal model by the Platycodon grandiflorum extract of the present invention:

Normal group;

Control group;

the experimental group treated with the Platycodon grandiflorum extract of the present invention at the concentration of 300 mg/kg (Platycodi radix extract 300 mg/kg, PGE-300 group) ;

the experimental group treated with the Platycodon grandiflorum extract of the present .invention at the concentration of 1000 mg/kg (Platycodi radix extract 1000 mg/kg, PGE-1000 group) ; and

the positive control group treated with the crude saponin extract at the concentration of 6 mg/kg (CS-6 group) .

Figure 8 is a diagram illustrating the clinical score of arthritis over the changes of paw thickness of the rheumatoid arthritis animal model by the Platycodon grandiflorum extract of the invention:

Normal group;

Control group;

the experimental group treated with the Platycodon grandiflorum extract of the present invention at the concentration of 100 mg/kg (Platycodi radix^' extract 100 mg/kg, 1

PGE-100 group);

the positive control group treated with the crude saponin extract at the concentration of 6 mg/kg (CS-6 group).

Figure 9 and Figure 10 are diagrams and picture respectively illustrating the changes in the front paw thickness of the rheumatoid arthritis animal model by the Platycodon grandiflorum extract of the invention:

Normal group; >

Control group;

the experimental group treated with the Platycodon grandiflorum extract of the present invention at the concentration of 300 mg/kg (Platycodi radix extract 300 mg/kg, PGE-300 group);

the experimental group treated with the Platycodon grandiflorum extract of the present invention at the concentration of 1000 mg/kg (Platycodi radix extract 1000 mg/kg, PGE-1000 group) ; and the positive control group treated with the crude saponin extract at the concentration of 6 mg/kg (CS-6 group) .

Figure 11 and Figure 12 are diagrams and picture respectively illustrating the changes in the hind paw thickness of the rheumatoid arthritis animal model by the Platycodon grandiflorum extract of the invention:

Normal group;

Control group;

Figure 13 is a diagram illustrating ; the effect of the Platycodon grandiflorum extract of the present invention on the levels of TNF-a and IL-Ιβ in the blood of the rheumatoid arthritis animal model:

Normal group;

Control group;

the experimental group treated with the platycodon grandiflorum extract of the present invention at the concentration of 100 mg/kg (Platycodi radix extract 100 mg/kg, PGE- 100 grou ) ;

[Best Mode]

Hereinafter, the present invention is described in detail.

The present invention provides a pharmaceutical composition for the prevention and treatment of rheumatoid arthritis comprising the Platycodon grandiflorum extract as an active ingredient.

The Platycodon grandiflorum extract is preferably prepared by the method composed of the following steps, but not always limited thereto:

1) extracting Platycodon grandiflorum using an extraction solvent;

2) filtering the extract obtained in step 1) ; and

3) concentrating the extract filtered in step 2) under reduced pressure to prepare the Platycodon grandiflorum extract.

In the method above, the Platycodon grandiflorum of step 1) is obtained by cultivation or purchased.

In the method above, the extraction solvent of step 1) is water, alcohol, or a mixed solvent of the same.

The alcohol herein is preferably Ci ~ C₂ lower alcohol, and the lower alcohol is preferably ethanol or methanol. The extraction method is one of the conventional methods accepted in this field, which is exemplified by the methods using an extraction apparatus such as hot-water extraction, soak extraction, supercritical extraction, subcritical extraction, high temperature extraction, high pressure extraction, reflux cooling extraction and ultrasonification extraction, and the methods using an adsorption resin such as such as XAD and HP-20. The extraction is preferably performed by reflux extraction with raising the extraction temperature or at room temperature, but not always limited thereto. The extraction solvent is preferably added to the dried Platycodon grandiflorum at the volume of 1 ~ 10 times the weight of the dried Platycodon grandiflorum and more preferably at the volume of 4 ~ 6 times the dried Platycodon grandiflorum. The extraction temperature is preferably 20 ~ 100°C, more preferably room temperature or 60 ~ 90°C, but not always limited thereto. The extraction time is preferably 3 - 36 hours, and more preferably 5 - 20 hours, but not always limited thereto. The extraction is preferably repeated 1 - 5 times, and more preferably 2 - 3 times, but not always limited thereto.

In the preparation method of the present invention, the concentration of the obtained extract is preferably performed by using a vacuum rotary evaporator, but not always limited thereto. The drying is preferably performed by reduced-pressurized drying, vacuum drying, boiling drying, spray drying, room temperature drying, or freeze drying, but not always limited thereto.

The Platycodon grandiflorum extract is preferably the Platycodon grandiflorum juice extract, but not always limited thereto.

The Platycodon grandiflorum juice extract is preferably prepared by the method comprising the following steps, but not always limited thereto:

1) performing juice extraction with the Platycodon grandiflorum, which is the primary extraction;

2) adding water to the Platycodon grandiflorum residue after the juice extraction in step 1) , followed by performing juice extraction, which is the secondary extraction;

3) mixing the juice extract obtained from the primary extraction of step 1) with the juice extract obtained from the secondary extraction of step 2), followed by centrifugation to eliminate the solid part.

The Platycodon grandiflorum juice extract preferably contains the enhanced contents of crude saponin and Platycodin, but not always limited thereto.

The Platycodin in this invention is preferably Platycodin D, but not always limited thereto. In general, Platycodin is mostly represented by Platycodin D, and Platycodin D2 , Platycodin D3 , and etc . sharing the similar structure are also included.

The Platycodon grandiflorum juice extract is preferably prepared by filtering the Platycodon grandiflorum extract with ultrafiltration membrane; and drying the filtrate to give the Platycodon grandiflorum juice extract with the molecular weight, of at least 1 , 000 , but not always limited thereto.

The Platycodon grandiflorum extract is preferably prepared by the method comprising the following steps, but not always limited thereto:

1 ) extracting Platycodon grandiflorum by using water, Ci ~ C₂ lower alcohol, or a mixed solvent thereof;

3 ) collecting the Platycodon grandiflorum extract wherein the sugar compound was separated in step 2 ) , followed by vacuum concentration and drying to prepare the Platycodon grandiflorum powder extract .

The alcohol herein is preferably Ci ~ C₂ lower alcohol, and the lower alcohol is preferably ethanol or methanol . The extraction method is one of the conventional methods accepted in this field, which is exemplified by the methods using an extraction apparatus such as hot-water extraction, soak extraction, supercritical extraction, subcritical extraction, high temperature extraction, high pressure extraction, reflux cooling extraction and ultrasonification extraction, and the methods using an adsorption resin such as such as XAD and HP- 20 . The extraction is preferably performed by reflux extraction with lb raising the extraction temperature or at room temperature, but not always limited thereto. The extraction solvent is preferably added to the dried Platycodon grandiflorum at the volume of 1 - 10 times the weight of the dried Platycodon grandiflorum arid more preferably at the volume of 4 ~ 6 times the dried Platycodon grandiflorum.. The extraction temperature is preferably 20 ~ 100°C, more preferably room temperature or 60 ~ 90°C, but not always limited thereto. The extraction time is preferably 3 ~ 36 hours, and more preferably 5 - 20 hours, but not always limited thereto. The extraction is preferably repeated 1 - 5 times, and more preferably 2 - 3 times, but not always limited thereto.

In step 3) , the ion exchange resin is preferably Diaion, Lewatit, Dowex, or Amberite, but not always limited thereto. The size exclusion chromatography uses sephadex fillers and preferably sephadex LH filler, but not always limited thereto. The ion exchange resin used in this invention was Diaion UBK, and precisely the resin was substituted into Na-form by using aqueous NaCl solution and then the NaCl was eliminated by using distilled water. .

The sugar compound separated above is glucose, fructose, sorbitol, sucrose, or monosaccharides.

In the method^" above, the filtrate passed through the ion exchange resin and the size exclusion chromatography in step 3) was dried to give the Platycodon grandiflorum extract having the molecular weight of at least 1,000, but not always limited thereto.

In the preparation method of the invention, the concentration of the obtained extract is preferably performed by using a vacuum rotary evaporator, but not always limited thereto. The drying is preferably performed by reduced-pressurized drying, vacuum drying, boiling drying, spray drying, room temperature drying, or freeze drying, but not always limited thereto.

The Platycodon grandiflorum extract preferably contains

Platycodin at least 1%.

The Platycodon grandiflorum extract prepared by the method of the invention preferably contains the enhanced effective saponin contents, and the effective saponin is Platycodin and more specifically Platycodin D.

In general, Platycodin is mostly represented by Platycodin D, and Platycodin D2 , Platycodin D3 , and etc. sharing the similar structure are also included. The present invention also provides a pharmaceutical composition for the prevention and treatment of rheumatoid arthritis comprising the Platycodon grandiflorum extract as an active ingredient.

2) adding water to the Platycodon grandiflorum residue after the juice extraction in step 1), followed by performing juice extraction, which is the secondary extraction;

3) mixing the juice extract obtained from the primary extraction of step 1) with the juice extract obtained from the secondary extraction of step 2) , followed by centrifugation to eliminate the solid part.

4) concentrating the juice extract from which the solid part was eliminated in step 3) , followed by separating the sugar compound by using ion exchange resin and the size exclusion chromatography;

5) spray-drying the juice extract wherein the sugar compound was separated in step 4) but not always limited thereto

The Platycodon grandiflorum juice extract preferably contains the increased contents of crude saponin and Platycodin, but not always limited thereto. The Platycodon grandiflorum extract preferably contains Platycodin at least 1% .

The Platycodin in this invention is preferably Platycodin D, but not always limited thereto. In general, Platycodin is mostly represented by Platycodin D, and Platycodin D2 , Platycodin D3 , and etc. sharing the similar structure are also included.

The Platycodon grandiflorum juice extract is preferably prepared by filtering the Platycodon grandiflorum extract with ultrafiltration membrane; and concentrating the filtrate under reduced pressure so that the Platycodon grandiflorum extract preferably has the molecular weight of at least 1,000, but not always limited thereto.

The present invention provides a health functional food for the prevention and improvement of rheumatoid arthritis comprising the Platycodon grandiflorum extract as an active ingredient . 1

The Platycodon grandiflorum extract preferably contains Platycodin at least 1%.

The Platycodin in this invention is preferably Platycodin D, but not always limited thereto. . In general, Platycodin is mostly represented by Platycodin D, and Platycodin D2, Platycodin D3 , and etc. sharing the similar structure are also included.

The present invention also provides a method for preparing the Platycodon grandiflorum extract having the increased content of effective saponin comprising the following steps :

1) extracting Platycodon grandiflorum by using water, Ci ~ C₂ lower alcohol, or a mixed solvent thereof;

2) concentrating the extract obtained in step 1) and separating the sugar compound from the extract by using ion exchange resin and size exclusion chromatography; and

3) collecting the Platycodon grandiflorum extract wherein the sugar compound was separated in step 2), followed by vacuum concentration and drying to prepare the Platycodon grandiflorum powder extract .

The sugar compound separated above is preferably glucose, fructose, sorbitol, sucrose, or a monosaccharide.

The effective saponin herein is preferably Platycodin, and more preferably Platycodin D.

In general, Platycodin is mostly represented by Platycodin

D, and Platycodin D2 , Platycodin D3 , and etc. sharing the similar structure are also included.

The Platycodon grandiflorum extract preferably contains Platycodin at least 1%. The present invention also provides a method for preparing the Platycodon grandiflorum extract having the increased content of effective saponin comprising the following steps:

3) mixing the juice extract^' obtained from the primary extraction of step 1) with the juice extract obtained from the secondary extraction of step 2) , followed by centrifugation to eliminate the solid part.

4) concentrating the juice extract from which the solid part was eliminated in step 3), followed by separating the sugar compound by using ion exchange resin and the size exclusion chromatography;

5) spray-drying the juice extract wherein the sugar compound was separated in step 4) .

The sugar compound separated above is preferably glucose, fructose, sorbitol, sucrose, or a monosaccharides.

In general, Platycodin is mostly represented by Platycodin D, and Platycodin D2 , Platycodin D3 , and etc. sharing the similar structure are also included.

The Platycodon grandiflorum extract preferably contains Platycodin at least 1%. The present invention also provides a pharmaceutical composition for the prevention and treatment of rheumatoid arthritis comprising the Platycodon grandiflorum extract prepared by the method above as an active ingredient.

The Platycodon grandiflorum extract preferably contains

Platycodin at least 1%. The Platycodin in this invention is preferably Platycodin D, but not always limited thereto. In general, Platycodin is mostly represented by Platycodin D, and Platycodin D2 , Platycodin D3 , and etc. sharing the similar structure are also included.

In general, the Platycodon grandiflorum extract contains Platycodin, the index ingredient saponin, approximately 0.1 ~ 0.2% when it is prepared by hot-water extraction, alcohol extraction, and juice extraction. The composition of other ingredients are as follows: crude saponin 0.3 ~ 2%, monosaccharides and disaccharides such as glucose, fructose, sorbitol, and sucrose, etc, 45 ~ 50%, fructooligosaccharide 30 ~ 40%, and the polysaccharide inulin 20 - 30%, which means the conventional extraction method is not good enough to prepare the Platycodon grandiflorum extract with the increased Platycodin content to 0.5 - II.

In this invention, the content of saponin including Platycodin in the Platycodon grandiflorum extract was increased by eliminating the sugar compound. It is better to eliminate the simple sugars such as glucose, fructose, sorbitol, and sucrose and other sugar compounds smaller than oligosaccharide from the Platycodon grandiflorum extract because they are the reason of physical unstability in the process of spray-drying for the mass -production . of the extract. Another method to increase the content of Platycodin, the method using a nano membrane separation has been reported. However, according to this method, monosaccharides and disaccharides including glucose, fructose, sorbitol, and sucrose are still included at the concentration of 40 ~ 50%, so that it is hard to increase the content of Platycodin up to 1%, even though the content of Platycodin can be increased up to 0.3% which is the double the level of the original Platycodin content in the Platycodon grandiflorum extract.

In a preferred embodiment of the present invention, the Platycodon grandiflorum extracts have been prepared by using various extraction solvents. The inventors also prepared the Platycodon grandiflorum juice extract via juice extraction and then measured the level of crude saponin in each extract. The filtrates filtered by ultrafiltration membrane were also collected and dried to prepare the Platycodon grandiflorum composition having the molecular weight of at least 1,000. Then, the changes in the content of crude saponin in the Platycodon grandiflorum juice extract were investigated. As a result, it was confirmed that the contents of polysaccharides and crude saponin were increased after the ultrafiltration twice as much as those in the Platycodon grandiflorum juice extract (Figure 1) .

500 ml of Diaion UBK was loaded in a glass tube (2.5 x 100 cm) and washed with 500 ml of distilled water, to which 200 ml of 5% aqueous NaCl solution was added to substitute thereof into Na-form. NaCl was eliminated by using 500^; ml of distilled water, followed by column chromatography. 2 g of the concentrated Platycodon grandiflorum hot-water extract containing Platycodin D at the concentration of 0.098% was dissolved in 20 ml of water, which was loaded in the column chromatography. 500 ml of distilled water was added thereto. The eluted materials were examined by TLC, during which the sugar compound was separated. After confirming the elimination of monosaccharides and disaccharides such as glucose, fructose, sorbitol, sucrose, and fructooligosaccharides , the remaining eluent proceeded to vacuum concentration. As a result, 210 mg of the compound was obtained. from the results of HPLC, it was confirmed that the content of Platycodin D in the Platycodon grandiflorum extract was increased approximately 10 times after the elimination of the sugar compound (see Table 2 and Figure 3) .

To investigate the therapeutic effect of the Platycodon grandiflorum extract of the present invention on rheumatoid arthritis, in vitro and in vivo experiments were performed. As a result, the Platycodon grandiflorum extract of the present invention was confirmed to have a significant therapeutic effect on rheumatoid arthritis. Particularly, the inventors treated the Platycodon grandiflorum extract to osteoclasts and observed the cells 4 days later. As a result, it was confirmed that the size and the differentiation of the osteoclasts were significantly reduced in the group treated with the Platycodon grandiflorum extract by 10 g/ml maximum, compared with the non- treated control group (see Figure 4) .

To investigate the effect of the Platycodon grandiflorum extract of the present invention on the rheumatoid arthritis animal model, the present inventors measured the changes of body weight from the adaption period to the arthritis induction period. result, as the administration dose of the

Platycodon grandiflorum extract was increased, the weight was increased significantly (see Figure To confirm the induction of rheumatoid arthritis, the levels of edema, erythema, and spasticity were calculated as clinical scores. As a result, after the first boosting in the groups treated with the Platycodon grandiflorum extract and in the groups treated with the crude saponin extract, the scores were similar to those of the normal group. After the second boosting, the clinical changes were observed and the clinical scores were lowered as the administration dose of the Platycodon grandiflorum extract was increased (see Figure 8).

To investigate the effect of the Platycodon grandiflorum extract of the present invention on the rheumatoid arthritis animal model, the inventors measured the paw thickness. After comparing the results, . it was confirmed that the paw thickness was significantly reduced in the. groups treated with the Platycodon grandiflorum extract and in the groups treated with the crude saponin (see Figure 9 ~ 12) . In the rheumatoid arthritis animal model, the levels of TNF-a (tumor necrosis factor-a) and IL-Ιβ (Interleukin 1β), the blood inflammation index factors, in each group were measured and compared. As a result, as the administration dose of the Platycodon grandiflorum extract was increased, the levels of the blood inflammation index factors were beginning to decrease or significantly reduced (see Figure 13) .

Therefore, the Platycodon grandiflorum extract of the present invention strongly induced the differentiation of osteoclasts and significantly reduced edema in the rheumatoid arthritis animal model, so that it could be effectively used as an active ingredient of a pharmaceutical composition for the prevention and treatment of rheumatoid arthritis . The composition comprising the Platycodon grandiflorum extract of the present invention can include, in addition to the Platycodon grandiflorum extract, one or more effective ingredients having the same or similar function to the Platycodon grandiflorum extract.

The composition of the present invention can additionally include a pharmaceutically acceptable additive, which is exemplified by starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, taffy, Arabia rubber, pregelatinized starch, corn starch, cellulose powder, hydroxypropyl cellulose, Opadry, sodium carboxy methyl starch, carunauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, dextrose, sorbitol, talc, etc. The pharmaceutically acceptable additive herein is preferably added by 0.1 ~ 90 weight part to the composition of the invention, but not always limited thereto.

The present invention also provides an enteric coated oral formulation comprising the Platycodon grandiflorum extract prepared by the method of the invention above as an active ingredient .

The Platycodon grandiflorum extract preferably contains Platycodin at least 1%. 2o

The composition of the present , invention can be administered orally or parenterally and be used in general forms of pharmaceutical formulation. And at this time, it is preferred for any formulation to be enteric-coated.

Particularly, pharmacokinetic test was performed with the Platycodin D (PD) included in the Platycodon grandiflorum extract of the present invention. From the results of the pharmacokinetic test, it was confirmed that the Platycodin D compound that was enteric-coated designed to release the drug in the intestines but not in the^' stomach was more efficient in minimizing the damage and side effects in the stomach than the Platycodin D compound that was administered orally and released in the stomach. Therefore, it was proved that the Platycodin D had to be enteric-coated in order to bring a significant therapeutic effect on rheumatoid arthritis.

The preferable composition for the enteric coating is as follows : mix

No. raw material function proportioning

(%)

primary

1 Platycodon grandiflorum extract 73.53

function

Hydroxypropyl methylCellulose 2^nd enteric

2 8.1

phthalate coating

3 cornstarch diluting agent 6.98

4 Lactose mixture diluting agent 3.37

NS 1050180000(Polyvinyl Alcohol

mixed agent)

(Polyvinyl Alcohol 40.0%

5 Titanium Dioxide 25.0% 3^rd coating 3.2

Calcium carbonate 17.5%

Hydroxypropyl methylCellulose 10.0%

Triethyl citrate 7.5%)

6 Calcium Carboxymethylcellulose disintegrator 1.16

7 silicon dioxide lubricant 1.16

8 glycerin plasticizer 0.9

9 Magnesium Stearate lubricant 0.8

Opadry NS Clear 121M190002

(Hydroxypropyl methylCellulose 1^st coating

10 0.5

90.091%

Glycerin 9.909%

11 Sodium carboxymethyl cellulose Gloss coating 0.16248

12 maltodextrin diluting agent 0.06237

13 glucose diluting agent 0.05085

14 lecithin diluting agent 0.0183

15 sodium citrate diluting agent 0.006

total 100.00

The effective dose of the composition of the present invention can be determined according to weight, age, gender, health condition, diet, administration frequency, administration method, excretion and severity of a disease. The dose is 0.001 ~ 200 nig/kg per day, and preferably 0.01 ~ 50 nig/kg per day, and administration frequency is preferably 1 - 6 times a day. The composition of the present invention can be administered alone or treated together with surgical operation, radiotherapy, hormone therapy, chemo- therapy and biological regulators .

.

The present invention provides a health functional food for the prevention and improvement of rheumatoid arthritis comprising the Platycodon grandiflorum extract prepared by the method above as an active ingredient .

The Platycodon grandiflorum extract preferably contains

Platycodin at least 1%.

The Platycodon grandiflorum extract of the present invention has a significant therapeutic effect on rheumatoid arthritis, so that it can be efficiently used as an active ingredient of a health functional food for the prevention and improvement of rheumatoid arthritis.

In this description, "health functional food" indicates the food produced with the supplement of such nutrients that are often lack in daily diet or raw materials or components (functional ingredients) having a useful function for human body. The purpose of such a health functional food is to maintain a normal function of human body or to maintain health by activating necessary physiological function, which has been approved by Ministry of Food and Drug Safety, but not always limited thereto, and any general health food can be included.

The Platycodon grandiflorum extract of the present invention can be used as a food additive. , In that case, the Platycodon grandiflorum extract of the present invention can be added as it is or as mixed with other food components according to the conventional method. The mixing ratio of active ingredients can be regulated according to the purpose of use (prevention or health enhancement). In general, to produce health food or beverages, the Platycodon grandiflorum extract of the present invention is added preferably by 0.01 ~ 90 weight part. However, if long term administration is required for health and hygiene or regulating health condition, the content can be lower than the above but higher content can be accepted as well since the Platycodon grandiflorum extract of the present invention has been proved to be very safe.

The health functional beverage composition of the present invention can additionally include various flavors or natural carbohydrates, etc, like other beverages in addition to the said compound. The natural carbohydrates above can be one of monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xilytole, sorbitol and erythritol. Besides, natural sweetening agents (thaumatin, stevia extract, for example rebaudioside A, glycyrrhizin, etc.) and synthetic sweetening agents (saccharin, aspartame, etc.) can be included as a sweetening agent. The content of the natural carbohydrate is preferably 1 ~ 20 g and more preferably 5 ~ 12 g in 100 g of the composition of the present invention. In addition to the ingredients mentioned above, the Platycodon grandiflorum extract of the present invention can include in variety of nutrients, vitamins, minerals (electrolytes), flavors including natural flavors and synthetic flavors, coloring agents and extenders (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acid, protective colloidal viscosifiers , pH regulators, stabilizers, antiseptics, glycerin, alcohols, carbonators which used to be added to soda, etc. The Platycodon grandiflorum extract of the present invention can also include natural fruit juice, fruit beverages and/or fruit flesh addable to vegetable beverages .

All the mentioned ingredients can be added singly or together. The mixing ratio of those ingredients does not matter in fact, but in general, each can be added by 0.1 ~ 20 weight part per 100 weight part of the Platycodon grandiflorum extract of the invention.

[Mode for Invention]

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 Platycodon grandiflorum (Platycodon grandiflorum A. DC) extract <!-!> Preparation of Platycodon grandiflorum water extract

1,000 g of Platycodon grandiflorum powder was mixed with 5,000 mi of distilled water, followed by reflux extraction twice at 90^°C for 6 hours. The obtained extract was cooled down, followed by centrifugation (10,000 x g) at room temperature for 30 minutes to eliminate solid parts. The solution separated by the centrifugation above was spray-dried. As a result, 368 g of Platycodon grandiflorum- water extract powder was obtained. <l-2> Preparation of Platycodon grandiflorum ethanol extract

1,000 g of Platycodon grandiflorum powder was mixed with 5,000 mi of ethanol, followed by reflux extraction twice in a water bath for 6 hours. The obtained extract was cooled down, followed by centrifugation (10,000 x g) at room temperature for 30 minutes to eliminate solid parts. The solution separated by the centrifugation above was vacuum-dried. As a result, 136 g of Platycodon grandiflorum ethanol extract powder was obtained.

<l-3> Preparation of Platycodon grandiflorum methanol extract

1,000 g of Platycodon grandiflorum powder was mixed with

5,000 mi of methanol, followed by reflux extraction twice in a water bath for 6 hours. The. obtained extract was cooled down, followed by centrifugation (10,000 x g) at room temperature for 30 minutes to eliminate solid parts. The solution separated by the centrifugation above was vacuum-dried. As a result, 205 g of Platycodon grandiflorum methanol extract powder was obtained.

<l-4> Preparation of Platycodon grandiflorum 90% ethanol extract · 1,000 g of Platycodon grandiflorum powder was mixed with 5,000 mi of ethanol/water solution (composed of 90% ethanol and 10% water), followed by reflux extraction twice in a water bath for 6 hours. The obtained extract was cooled down, followed by centrifugation (10,000 x g) at room temperature for 30 minutes to eliminate solid parts. The solution separated by the centrifugation above was vacuum-dried. As a result, 156 g of Platycodon grandiflorum 90% ethanol extract powder was obtained. <l-5> Preparation of Platycodon grandiflorum 75% ethanol extract

1,000 g of Platycodon grandiflorum powder was mixed with 5,000 mi of ethanol/water solution (composed of 75% ethanol and 25% water), followed by reflux extraction twice in a water bath for 6 hours. The obtained extract was cooled down, followed by centrifugation (10,000 x g) at room temperature for 30 minutes to eliminate solid parts. The solution separated by the centrifugation above was vacuum-dried. As a result, 175 g of Platycodon grandiflorum 75% ethanol extract powder was obtained.

<l-6> Preparation of Platycodon grandiflorum 50% ethanol extract

1,000 g of Platycodon grandiflorum powder was mixed with 5,000 mi of ethanol/water solution (composed of 50% ethanol and 50% water) , followed by reflux extraction twice in a water bath for 6 hours. The obtained extract was cooled down, followed by centrifugation (10,000 x g) at room temperature for 30 minutes to eliminate solid parts. The solution separated by the centrifugation above was vacuum-dried. As a result, 230 g of Platycodon grandiflorum 50% ethanol extract powder was obtained.

<l-7> Preparation of Platycodon grandiflorum 25% ethanol extract

1,000 g of Platycodon grandiflorum powder was mixed with

5,000 mi of ethanol/water solution (composed of 25% ethanol and 75% water) , followed by reflux extraction twice in a water bath for 6 hours. The obtained extract was cooled down, followed by centrifugation (10,000 x g) at room temperature for 30 minutes to eliminate solid parts. The solution separated by the centrifugation above was vacuum-dried. As a result, 305 g of Platycodon grandiflorum 25% ethanol extract powder was obtained.

<l-8> Preparation of Platycodon grandiflorum 10% ethanol extract

1,000 g of Platycodon grandiflorum powder was mixed with 5,000 mi of ethanol/water solution (composed of 10% ethanol and 90% water), followed by reflux extraction twice in a water bath for 6 hours. The obtained extract was cooled down, followed by centrifugation (10,000 x g) at room temperature for 30 minutes to eliminate solid parts. The solution separated by the centrifugation above was vacuum-dried. As a result, 356 g of Platycodon grandiflorum 10% ethanol extract powder was obtained. <l-9> Preparation of Platycodon grandiflorum juice extract

The primary juice extraction was performed with 5,000 g of fresh Platycodon grandiflorum and as a result 300 mi of Platycodon grandiflorum fresh juice was obtained. The remaining Platycodon grandiflorum, after the primary juice extraction, was added with 1,000 mi of water, followed by the secondary juice extraction at room temperature with stirring for 4 ~ 10 hours. As a result, 1,100 mi of Platycodon grandiflorum juice was obtained. The Platycodon grandiflorum fresh juice juices obtained from the primary and the secondary juice extraction were mixed together, followed by centrifugation at 10,000 x g for 30 minutes to eliminate solid parts. The solution separated by the centrifugation above was spray-dried. As a result, 476 g of Platycodon grandiflorum juice extract powder was obtained.

Example 2 : Separation of crude saponin from Platycodon grandiflorum extract

<2-l> Separation of crude saponin from Platycodon grandiflorum water extract

Crude saponin was separated from the Platycodon grandiflorum water extract prepared in Example 1 by the method below.

Particularly, 100 g of the Platycodon grandiflorum water extract was dissolved in 1,000 ml of water, which was loaded in the column (50 x 250 mm) filled with 500 mi of reverse phase gel (HP-20, RP-18, or MCI gel) to make crude saponin attached thereon. To eliminate the sugar compounds (glucose, sorbitol, fructose, sucrose, and inulin including fructooligosaccharide) which were not attached on the column, 1,000 mi of water and 500 mi of 3 ~ 5% aqueous acetonitrile solution were flowed over the column continuously. To eliminate acetonitrile, 500 mi of water was flowed over the column again. Once the sugar compounds were completely eliminated, 30 ~ 90% aqueous ethanol solution (500 mi) was flowed over the column enough to detach the attached components. The obtained aqueous ethanol solution was vacuum- concentrated and as a result 7 g of solid part was obtained. 50 mi of ethanol was added to the obtained solid part and the solid part still remaining non-dissolved in the ethanol was filtered out. 100 mi of ethyl acetate was added to the resulting filtrate. The produced solid was filtered and dried well. As a result, 6.5 g of crude saponin was obtained.

<2-2> Separation of crude saponin from Platycodon grandiflorum juice extract

Crude saponin was separated from 100 g of the Platycodon grandiflorum juice extract prepared in Example 1 and purified by the same manner as described in Example <2-l>. As a result,

14.8 g of crude saponin was obtained.

,

Example 3; Preparation of the composition comprising Platycodon grandiflorum saponin via filtration using ultra- filtration membrane

The ultra-filtration membrane used herein was Pellicon 2 TFF system (Millipore USA, PART# xi42 pmOOl) . 100 g of the Platycodon grandiflorum extract prepared in Example 1 was dissolved in 18,000 mi of distilled water, which was filtered with ultra-filtration membrane (Pellicon 2, 1 KDa) until the residue became 100 mi. 1,000 mi of water was added to the remaining residue, which was also filtered with ultra- filtration membrane (Pellicon 2, 1 KDa) until the volume of the residue became 100 mi or less. The filtrate was collected and concentrated under reduced pressure. As a result, the Platycodon grandiflorum composition having the molecular weight at least 1,000 was obtained as shown in Table 1 below.

[Table l]

Example 4: Preparation of the Platycodon grandiflorum extract comprising Platycodin at a high concentration

To increase the concentration of Platycodin D, the inventors eliminated monosaccharides and disaccharides such as glucose, fructose, sorbitol, and sucrose and fructooligosaccharide by using ion exchange resin such as Diaion, Lewatit, Dowex, and Amberite and size exclusion chromatography. Particularly, 500 mi of Diaion UBK was loaded in a glass tube (2.5 x 100 cm) and washed with 500 mi of distilled water, to which 200 ml of 5% aqueous NaCl solution was added in order to substitute thereof into Na-form. NaCl was eliminated by us.ing 500 ml of distilled water, leading to the preparation of column chromatography. 2 g of the concentrated Platycodon grandiflorum hot-water extract (PD content: 0.098%) prepared in Example 1 was dissolved in 20 ml of water, which was slowly loaded in the column chromatography. At least 500 ml of distilled water was added thereto. The eluted materials were examined by TLC, during which the sugar compound was separated. After confirming the elimination of monosaccharides and disaccharides such as glucose, fructose, sorbitol, sucrose, and fructooligosaccharides , the remaining eluent proceeded to vacuum concentration. As a result, 210 mg of the compound was obtained.

As shown in Table 2 and Figure 3 , the prepared compound was examined by HPLC. As a result, it was confirmed that the content of Platycodin D in the Platycodon grandiflorum extract was 10 times increased after eliminating the sugar compounds (Table 2 and Figure 3).

[Table 2]

Component PD

Retention time (min) 64.382

Platycodon

Area (uVsec) ) 2564056.54 grandiflorum extract

(before the Area% label (%) 100.000 BMB elimination of sugar

compound) Content 381.448

Component PD

Retention time (min) 64.882

Platycodon Area (uVsec) ) 34210565.32 grandifloru extract Area% label (%) 100.000 BMB

(after the

elimination of sugar

compound) Content 3887.236

Experimental Example 1: Investigation of polysaccharides and crude saponin contents

To investigate the contents of polysaccharides and crude saponin in the Platycodon grandiflorum extract using ultrafiltration, solvent delivery pump (NS-4000i Futecs Co., Ltd.) was used and the column for the standard analysis was OH pak (SB-802, 802.5, 806M) or its equivalent one. The temperature of the column was maintained at 60^°C and the detector was Refractive Index detector (IR-101 shodex) . The flow rate was 0.9 ni£/min and the eluent was distilled water. The total analysis time was 100 minutes.

As a result, as shown in Figure 1, after the juice extraction, the materials with the molecular weight of up to 1,000 took a larger part than the materials with the molecular weight of at least 2,000 in the Platycodon grandiflorum juice extract. However_/ after the ultrafiltration, the materials with the. molecular weight of at least 2,000 took a larger part.

From the results above, it was confirmed that the contents of polysaccharides and crude saponin were increased at least double after the ultrafiltration, compared with the contents of them before the ultrafiltration (Figure 1) .

Experimental Example 2: Inhibitory effect of the Platycodon grandiflorum extract on the osteoclast differentiation in rheumatoid arthritis

It has been reported that the activation of the differentiation of osteoclasts causes the joint destruction in rheumatoid arthritis. In this invention, the inhibitory effect of the Platycodon grandiflorum extract of the invention prepared in Example 4 on the differentiation of osteoclasts according to the concentration was investigated in order to confirm the efficacy of the same.

Particularly, bone marrow cells that could be possibly differentiated into osteoclasts were separated. For the culture of osteoclasts, PBMC (4 x 10⁵ cell/well) and RASF (0.5 x 10³ cell/well) were co-cultured for two weeks and. then CD40L+ or CD40L- cells (0.5 x 10⁴ cell/well) or T cells (10⁴ cell/well) were distributed in a 48 well plate, which were treated with 30 ng/ml of M-CSF and 150 ng/ml of RANKL, followed by culture for 4 days. The cultured cells were treated respectively with the control, ethanol control, and the Platycodon grandiflorum extract of the invention prepared in Example 4 at the concentrations of 2, 5, and 10 g/ml . On the 4^th day of culture, TRAP staining was performed. Then, the . cells were analyzed by using image analysis system (Image Gauge, Tokyo, Japan).

As shown in Figure 4, the osteoclasts were observed 4 days after the treatment of the Platycodon grandiflorum extract of the invention. As a result, it was confirmed that the size and the differentiation of osteoclasts were reduced in the group treated with the Platycodon grandiflorum extract at the concentration of 10 βg/ml maximum 4 days after the treatment, compared with the control not-treated with the extract (Figure 4) . Therefore, it was confirmed that the extract inhibited the differentiation of osteoclasts so that it could be efficiently used for the treatment of rheumatoid arthritis.

Experimental Example 3: Improvement of rheumatoid arthritis by the Platycodon grandiflorum extract in the rheumatoid arthritis animal model

<3-l> Preparation of experimental animal

For the experimental animal, specific-pathogen free (SPF) male DBA/1 mice at 7 weeks were distributed from Samtaco BioKorea (Osan City, Korea) . The mice had one week of adaptation period before being used for experiment. General solid feeds (Samtaka Gyunggi, Korea) were provided as a diet and filtered drinking water was filled up every day for free taking. The temperature during the experimental period was maintained at 23±1^°C, the humidity was maintained at 50±5%, and the noise level was controlled below 60 phone. The light hours were 08:00 ~ 20:00 (12 hours a day) and the intensity of illumination was controlled at 150 ~ 300 Lux. Air circulation was performed 10 ~ 12 times per hour. The present inventors complied the guide for the care and use of laboratory animals set by Wonkwang University.

The experimental animals after the adaptation period were grouped by Z method (randomized block design) . The experimental groups were the normal group, the control group induced with rheumatoid arthritis (RA) , the group induced with rheumatoid arthritis (RA) and then treated with 100 mg/kg of the Platycodon grandiflorum extract of the present invention prepared in Example 4, the group induced with RA and treated with 300 mg/kg of the Platycodon grandiflorum extract, the group induce with RA and then treated with 1,000 mg/kg of the PIatycodon grandiflorum extract of the present invention, and the group induced with RA and treated with 6 mg/kg of the crude saponin separated in Example 2. Each group was composed of 10 mice. The body weight and feeds . and drinking water that they took were all measured once a week. To calculate the amount of feeds and drinking water that they took, the remaining amounts of feeds and drinking water after every supply were measured at a set time.

As shown in the schematic diagram of Figure 5, rheumatoid arthritis was induced by using bovine type II collagen. Particularly, 2 mg/m# of bovine type IT collagen solution was mixed with the equal amount of complete Freund' s adjuvant (CFA, Chondrex, Redmond, A, USA) . On day 0, 150 βί of the mixture solution (100 βg of type II collagen) was slowly injected intradermally into the region under 1.5 ~ 3 cm from the basal part of the tail to induce rheumatoid arthritis primarily. On day 21, bovine type II collagen solution was mixed with incomplete Freund' s adjuvant (IFA, Chondrex), likewise as shown in the primary induction. The mixture was slowly injected into the basal part of the tail to induce joint destruction, deformity, and edema. The samples for each group were orally treated from the administration of the immunostimulant on day 21, every day via oral administration, and the control group was administered with an equal amount of drinking water. The thickness of paw was measured every week. On day 40, and the autopsy of the experimental animals was performed. Then, each biomarker was analyzed.

<3-2> Changes in the body weight and the amount of diet/drinking water

To investigate the effect of the Platycodon grandifloru extract of the present invention prepared in Example 4 on the rheumatoid arthritis (RA) animal model prepared in Experimental Example <3-l>, changes in the body weight of each experimental group were measured during the experimental period from the adaptation period.

Compared with the normal group, the body weight of the control group was reduced according to the administration of the RA inducing material. The body weight began to decrease from the first boosting. The body weight continued to decrease from the second boosting over the time. On day 30, the body weight was reduced, compared with the early body weight, due to the rheumatoid arthritis pain. After day 33, the body weight decrease was not continued any longer and remained as the same. In the meantime, unlike the control group, the body weight of the group treated with the Platycodon grandiflorum extract of the present invention (Platycodi radix extract group) was similar to that of the normal group after the administration of the RA inducing material. After the first boosting, the body weight was reduced and after the second boosting the lowest body weight was observed, but on day 33 the body weight began to be recovered.

On day 36 when the experiment ended, the body weight of each group was compared. As a result, the body weight of the normal group was 22.6 ± 0.4 g, the body weight of the control group was 19.0 + 0.3 g, there was a slight difference between these two groups. And the body weight of the group treated with 100 mg/kg of the Platycodon grandiflorum extract (Platycodi radix extract 100 mg/kg, PGE-100 group) was 19.8 ± 0.4 g, the group treated with 300 mg/kg of the Platycodon grandiflorum extract (Platycodi radix extract 300 mg/kg, PGE-300 group) was 19.9 ± 0.2 g, and the group treated with 1,000 mg/kg of the Platycodon grandiflorum extract (Platycodi radix extract 1000 mg/kg, PGE-1000 group) was 21.1 ± 0.6 g, showing that as the concentration of the sample increased, the body weight was significantly increased dose-dependently. In the meantime, the body weight of the positive control group (crude saponin extract 6 mg/kg, CS-6 group) was 20.1 + 0.4 g which was higher than those of the control group and the groups of PGE-100 and PGE-300, but was lower than that of the PGE-1000 group. Therefore, it was confirmed that the body weight of the group treated with 1000 mg/kg of the Platycodon grandiflorum extract was the highest (Figure 6) .

As shown in Figure 7 , there was a slight difference in daytime diet and drinking water among the experimental groups during the rheumatoid arthritis experimental period, but that difference was not dose-dependent nor significant (Figure 7) .

<3-3> Clinical observation

To confirm the induction of rheumatoid arthritis, the levels of edema, erythema, and spasticity were measured and converted into the numbers from 0 to 3, which would be the rheumatoid arthritis cLinical scores . When swelling and pathological findings were not observed, the point was 0, when light edema and erythema were observed, the point was 1, when edema was clearly observed, the point was 2, and when edema and spasticity were seriously developed, the point was 3. The rheumatoid arthritis clinical scores for each group were averaged by individual scores .

As shown in Figure 8, the clinical scores for edema in both the front paw and the hind feet paw were significantly increased in the control group after the first boosting and the second boosting, compared with the normal group. The clinical score was most significantly increased on day 30 after the second boosting, compared with the original condition. In the meantime, the groups treated with the Platycodon grandiflorum extract and crude saponin showed equal clinical scores to that of the normal group after the first boosting. After the second boosting, a clinical change was observed but the clinical scores were still lower or similar to that of the normal group, compared with the control group. On day 36, the clinical scores for the changes in paw for each group were as follows: the normal group: 0.56 ± 0.24, the control group: 4.00 ± 0.00, PGE-100 group: 3.78 ± 0.15, PGE-300 group: 3.33 ± 0.33, PGE-1000 group: 2.67 ± 0.33, and CS- 6 group: 2.78 ± 0.32. As the administration concentration of the Platycodon grandiflorum extract was increased, the clinical scores were significantly lowered (Figure 8) .

<3-4> Confirmation of the changes of edema in the front/hind paw of rheumatoid arthritis animal model

To investigate the effect of the Platycodon grandiflorum extract prepared in Example 4 of the present invention on the rheumatoid arthritis (RA) animal model, the paw thickness of the animal was measured and compared.

As shown in Figures 9 - 12, the thickness of the front paw of each group was as follows: the normal group: 0.19 ± 0.01 cm, and the control group: 0.34 ± 0.01 cm, showing that the paw thickness was significantly increased by the induction of arthritis. The thickness of the front paw of each group treated with the Platycodon grandiflorum extract of the present invention was as follows: PGE-100 group: 0.35 ± 0.01 cm, PGE-300 group: 0.34 ± 0.01 cm, PGE-1000 group: 0.29 ± 0.02 cm, and CS group (positive control): 0.31 ± 0.01 cm. Therefore, they displayed a significant difference compared with the control group (Figure 9a) . The front paw thickness index of each group were calculated by the change of paw thickness compared with the early paw thickness. As a result, the normal group was 102.95 ± 4.46%, the control group was 185.31 ± 2.93%, PGE-100 group was 190.26 ± 3.49%, PGE-300 group was 176.10 ± 1.86%, PGE-1000 group was 151.29 ± 6.89%, and CS group was 161.38 ± 5.97%. Therefore, the increase of the paw thickness of the experimental group induced by rheumatoid arthritis was suppressed or significantly suppressed by the Platycodon grandiflorum extract of the present invention compared with the control group (Figure 9b). The changes in the front paw of each experimental group in the rheumatoid arthritis animal model were observed. As a result, the paw thickness of the groups treated with the Platycodon grandiflorum extract and crude saponin of the present invention were significantly reduced, confirming the effect of the Platycodon grandiflorum extract and crude saponin (Figure 10) .

As for the hind paw, the paw thickness of each group was as follows: the normal group: 0.25 ± 0.00 cm, the control group: 0.31 ± 0.01 cm, PGE-100 group: 0.30 ± 0.02 cm, PGE-300 group: 0.28 ± 0.01 cm, PGE-1000 group: 0.26 ± 0.00 cm, and CS group: 0.28 + 0.01 cm, showing that the thickness of the hind paw of the groups treated with the Platycodon grandiflorum extract and crude saponin of the present invention were all reduced (Figure 11a) . The changing index of the paw thickness in each group compared with the early paw thickness was as follows : the normal group: 103.33 ± 3.12%, and the control group: 129.12 ± 3.81%, indicating the paw thickness was increased after the induction of arthritis . The changing index of the paw thickness in each experimental group was as follows: PGE-100 group: 121.40 ± 5.52%, PGE-300 group: 115.40 ± 3.87%, PGE-1000 group: 109.87 ± 1.77%, and CS group: 113.36 ± 2.23%, showing that the paw thickness of each experimental group treated with the sample of the present invention was significantly reduced, compared with that of the control group (Figure lib) . The changes in the hind paw of each group in RA animal model were also observed. As a result, it was confirmed that the hind paw thickness was significantly reduced in the groups treated with the Platycodon grandiflorum extract and crude saponin of the present invention (Figure 12) .

<3-5> Analysis of blood index

The levels of TNF- (tumor necrosis factor-a) and IL-Ιβ

(Interleukin 1β), the blood inflammation indexes, in the rheumatoid arthritis animal model were analyzed and compared among the experimental groups. Blood was drawn from the abdominal vein and loaded in a tube, followed by centrif gation at 3000 rpm for 10 minutes to isolate serum. The separated serum alone was used for the analysis. TNF-a and IL-Ιβ were analyzed with the serum, which was performed at Green Cross Laboratories (GC Labs, Korea) .

As shown in Figure 13, the level of TNF- in blood was 67.97 ± 7.75 ug/ml in the normal group and 97.73 ± 6.52 ug/ml in the control group, showing that the content was significantly increased in the experimental group in the rheumatoid arthritis model. In the meantime, the TNF-a content in blood was gradually reduced in the group treated with the Platycodon grandiflorum extract of the present invention and the level in PGE-100 group was 81.34 ± 5.52 ug/ml, in PGE- 300 group was 73.28 ± 3.76 ug/ml, in PGE- 1000 group was 63.30 ± 2.68 ug/ml, showing that the content in blood was reduced or significantly reduced as the concentration of the Platycodon grandiflorum extract increased, compared with that of the control . The content in CS group, the positive control, was 63.61 ± 2.73 ug/ml, which was similar to that of PGE-1000 group (Figure 13a) . The level of IL-Ιβ in blood was also compared among the experimental groups. As a result, the content was 98.12 + 8.50 ug/ml in the normal group and 157.65 ± 16.10 ug/ml in the control group, showing that the content in the control group was significantly increased. In the meantime, the level of blood IL-Ιβ was reduced in the experimental group treated with the Platycodon grandiflorum extract dose-dependently and as a result, the level in PGE-100 group was 130.82 ± 8.90 ug/ml, the level in PGE-300 group was 112.29 ± 4.48 ug/ml, and the level in PGE-1000 group was 105.56 ± 5.97 ug/ml. The level in CS group was 106.21 ± 6.48 ug/ml, which was similar to that of PGE -1000 group (Figure 13b) .

Experimental Example 4 : Pharmacokinetic test

<4-l> Selection and preparation of test animal

Male SD rats (190 ~ 215 g) at the age of 6 - 8 weeks purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. were used as the test animal. The SD rats were divided into 4 groups randomly based on the body weight (group 1 ~ group 4) . Each group was composed of 6 rats. A catheter was inserted in the right jugular vein of every rat. The group 1 rat which was supposed to get intravenous administration was inserted with a catheter in the femoral vein. The small intestine of the group 3 rat supposed to get intra-duodenal administration was exposed via the midline abdominal incision and the stomach was cut a little trough which a polyethylene tube was inserted. The small intestine of the group 4 rat supposed to get intra- ileal administration was exposed via the midline abdominal incision and the ileum was cut a little trough which a polyethylene tube was inserted. The exposed end was covered by a lid so that the duodenal and ileal contents or compounds were not emitted. After the surgery, the rats had 1 week recovery period.

<4-2> Administration of Platycodin D for the pharmacokinetic test

Platycodin D (PD) was administered to the test animals prepared in Example <4-l> for the pharmacokinetic test.

Particularly, the group 1 was the control group administered with PD via intravenous injection. Before the pharmacokinetic test, the SD rats were fasted for 16 hours and PD was administered at the single unit dose (5 mg/kg) via the femoral vein catheter. After the administration, 200 μί of blood sample was collected through the jugular vein catheter from the group administered with PD via intravenous injection at the time point of 0 min., 30 min., 1 nr., 2 nr., 4 hr. , and 6 nr. from the administration. The blood sample was collected in a sample tube containing EDTA, followed by centrifugation at 4°C for 5 minutes at 4000 rpm. The obtained plasma was transferred into another sample tube and stored at -20^°C .

The group 2 was the group orally administered with PD . Before the pharmacokinetic test, the SD rats were fasted for 16 hours and then administered orally with PD at the single unit dose of 10 mg/kg. After the administration, 200 μί of blood sample was collected through the jugular vein catheter from the group administered with PD via oral administration at the time point of 0 min., 30 min., 1 hr., 2 hr., 4 hr., and 6 hr. from the administration. The blood sample was collected in a sample tube containing EDTA, followed by centrifugation at 4^°C for 5 minutes at 4000 rpm. The obtained plasma was transferred into another sample tube and stored at -20^°C .

The group 3 was the group treated with PD via intra- duodenal administration. Before the pharmacokinetic test, the SD rats were fasted for 16 hours and then administered with PD at the single unit dose of 10 mg/kg via intra-duodenal administration. After the administration, 200 βί of blood sample was collected through the jugular vein catheter from the group administered with PD via intra-duodenal administration at the time point of 0 min., 30 min. , 1 hr., 2 hr. , 4 hr. , and 6 hr. from the administration. The blood sample was collected in a sample tube containing EDTA, followed by centrifugation at 4°C for 5 minutes at 4000 rpm. The obtained plasma was transferred into another sample tube and stored at -20 ^°C.

The group 4 was the grou treated with PD via intra-ileal administration. Before the pharmacokinetic test, the SD rats were fasted for 16 hours and then administered with PD at the single unit dose of 10 mg/kg via intra- ileal administration. After the administration, 200 μί of blood sample was collected through the jugular vein catheter from the group administered with PD via intra- ileal administration at the time point of 0 min. , 30 min. , 1 hr. , 2 hr. , 4 hr . , and 6 hr. from the administration. The blood sample was collected in a sample tube containing EDTA, followed by centrifugation at 4°C for 5 minutes at 4000 rpm. The obtained plasma was transferred into another sample tube and stored at -20 ^°C .

In addition, urine sample was taken from the rat of each group. Before the administration of PD, urine was forced by abdominal compression. Two hours after the administration of PD, urine was collected using a metabolic cage with care so that it was not contaminated with feces. The urine sample was centrifuged at 4 °C for 5 minutes at 4000 rpm. The supernatant was transferred into another sample tube and stored at -20^°C. <4-3> Analysis of the pharmacokinetic test results

To measure the concentrations of PD in the plasma and urine samples collected in Example <4-2>, the samples were pre -treated with a proper organic solvent, followed by LC-MS/MS analysis.

Particularly, before performing LC-MS/MS analysis with the plasma and urine samples obtained in Example 3, the samples were purified by using ZipTips TM (Millipore) according to the manufacturer's protocol. Then, the purified samples were analyzed by using Synapt High Definition mass spectrometer (Waters, Manchester, UK) (Jang et al . , 2009, Oncogene 28: 1529- 1536; Kim et al . , 2012, Stem Cells Dev. 10.1089/scd .2011.0243 ) .

As a result, it was confirmed that the time dependent PD concentration in the plasma over the administration method was as shown in Figure 2 (Figure 2) .

The pharmacokinetic indexes for the analysis of in vivo pharmacokinetic patterns according to the administration method were as shown in Table 3.

[Table 3]

As shown in Figure 2 and Table 3, Tmax (time to the maximum concentration in plasma) was 0.5 hr and not different among the groups but Cmax (maximum concentration index in plasma) was 4.8 times and 12.7 times higher respectively in the intra-duodenal administration group and the intra-ileal administration group than the oral administration group. That showed that when PD was administered directly through the duodenum and the ileum, it was better absorbed in plasma. That is, the direct administration of PD through the duodenum or the ileum could reduce the total amount of administration, compared with the oral administration, and it was also expected that the side effects accompanied by the oral administration such as abdominal inflation or discomfort could be reduced.

AUC value (area value in the blood concentration - time graph that is the index closely related to the in vivo bioavailability of an administered compound) up to 24 hours after the intravenous injection of PD was 6.5, while AUC up to 24 hours after the oral administration was 0.04. AUC after the intra-duodenal administration and the intra-ileal administration was 0.14 and 0.38 respectively. Therefore, it was confirmed that AUC value was 3.5 times and 9.5 times higher respectively via the intra-duodenal administration and the intra- ileal administration than the value of the oral administration, showing that the in vivo bioavailability of PD was increased more efficiently by intra-duodenal administration or intra- ileal administration than by oral administration.

Bioavailability (F24h, %) was 0.6 by oral administration, 2.2 by intra-duodenal administration, and 5.9 by intra- ileal administration, which were comparatively lower than that of intravenous injection. But the bioavailability of intra- duodenal administration and of intra-ileal administration was 3.7 times, and 9.8 times higher respectively than that of oral administration. That is, when PD was administered by intra- duodenal administration or intra-ileal administration, the pharmacological effect was greater than when PD was administered by oral administration.

From the above analyzed pharmacokinetic indexes, it was confirmed that when PD is administered as coated with a special enteric coating so that it does not melt in the stomach and instead is released in the intestines, it can maximize a pharmaceutical effect with a minimum dose with reducing damage and side effects in the stomach.

Those skilled in the art will appreciate that the conceptions 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

[ CLAIMS ]

[Claim l]

A pharmaceutical composition for the prevention and treatment of rheumatoid arthritis comprising the Platycodon grandiflorum extract as an active ingredient.

[Claim 2]

The pharmaceutical composition for the prevention and treatment of rheumatoid arthritis according to claim 1, wherein the Platycodon grandiflorum extract is extracted by using water, Ci ~ C₂ lower alcohol or a mixed solvent thereof,

[Claim 3]

The pharmaceutical composition for the prevention and treatment of rheumatoid arthritis according to claim 1, wherein the lower alcohol is ethanol or methanol.

[Claim 4]

The pharmaceutical composition for the prevention and treatment of rheumatoid arthritis according to claim 1, wherein the Platycodon grandiflorum extract is prepared by the method comprising the following steps:

2) adding water to the Platycodon grandiflorum after the juice extraction in step 1) , followed by performing juice extraction, which is the secondary extraction;

3) mixing the juice extract obtained from the primary extraction of step 1) with the juice extract obtained from the secondary extraction of step 2 )' , followed by centrifugation to eliminate the solid part;

4) filtering the juice extract after eliminating the solid part in step 3) with ultrafiltration membrane, followed by spray-drying.

[Claim 5]

The pharmaceutical composition for the prevention and treatment of rheumatoid arthritis according to claim 1 , wherein the Platycodon grandiflorum extract is prepared by the method comprising the following steps:

1 ) extracting Platycodon grandiflorum by using water, Ci ~ C₂ lower alcohol , or a mixed solvent thereof

3) collecting the Platycodon grandiflorum extract wherein the sugar compound was separated in step 2 ) , followed by vacuum concentration and drying to prepare the Platycodon grandiflorum powder extract.

[Claim 6]

1 ) performing juice extraction with the Platycodon grandiflorum, which is the primary extraction;

2 ) adding water to the Platycodon grandiflorum residue after the juice extraction in step 1) , followed by performing juice extraction, which is the secondary extraction;

[Claim 7]

The pharmaceutical composition for the prevention and treatment of rheumatoid arthritis according to any of claim 1 to claim 6, wherein the Platycodon grandifloru extract contains Platycodin at least 1%. [Claim 8]

A health functional food for the prevention and improvement of rheumatoid arthritis comprising the Platycodon grandiflorum extract as an active ingredient. [Claim 9]

The health functional food for the prevention and improvement of rheumatoid arthritis according to claim 8, wherein the Platycodon grandiflorum extract contains Platycodin at least 1% . [Claim 10]

An enteric coated oral formulation comprising the Platycodon grandiflorum extract.

■ 5

[Claim ll]

The enteric coated oral formulation according to claim 10, wherein the enteric coating is performed by the following steps:

1) primary coating with the primary coating agent at flow 10 rate of 1 -25 ml/min for 5 - 30 minutes;

2) secondary coating with the secondary coating agent at flow rate of 30 - 60 ml/min for 30 - 50 minutes; and

3) tertiary coating with the tertiary coating agent at flow rate of 1 - 25 ml/min for 5 - 20 minutes.

15 ^"

[Claim 12]

The enteric coated oral formulation according to claim 11, wherein the primary coating agent is the mixture of hydroxypropyl methylcellulose and glycerin.

^• 20

[Claim 13]

The enteric coated oral formulation according to claim 11, wherein the secondary coating agent is hydroxypropyl methylcellulose phthalate .

2

[Claim 14]

The enteric coated oral formulation according to claim 11, wherein the tertiary coating agent is the mixture of polyvinyl alcohol, titanium dioxide, calcium carbonate, hydroxypropyl thylcellulose , and triethyl citrate

[Claim 15 ]

The enteric coated oral formulation according to claim 10 , wherein the oral formulation additionally contains one or more compounds selected from the group consisting of excipients, disintegrating agents, lubricants, and plasticizers .

[Claim 16 ]

The enteric coated oral formulation according to claim 10 , wherein the enteric coated oral formulation is used for the prevention and treatment of rheumatoid arthritis.

[Claim 17 ]

A method for preparing the Platycodon grandiflorum extract with enhanced effective saponin contents comprising the following steps:

1 ) extracting Platycodon grandiflorum by using water, Ci ~

C₂ lower alcohol, or a mixed solvent thereof;

3) collecting the Platycodon grandiflorum extract wherein the sugar compound was separated in step 2 ) , followed by vacuum concentration and drying to prepare the Platycodon grandiflorum powder extract .

[Claim 18 ]

[Claim 19]

The method for preparing the Platycodon grandiflorum extract according to claim 17 or claim 18, wherein the sugar compound is glucose, fructose, sorbitol, sucrose, or monosaccharide . [Claim 20]

The method for preparing the Platycodon grandiflorum extract according to claim 17 or claim 18, wherein the effective saponin is Platycodin. [Claim 2l]

The method for preparing the Platycodon grandiflorum extract according to claim 17 or claim 18, wherein the Platycodon grandiflorum extract contains Platycodin at least 1%.

[Claim 22]

A pharmaceutical composition for the prevention and treatment of rheumatoid arthritis comprising the Platycodon grandiflorum extract prepared by the method of claim 17 or claim 18 as an active ingredient.

[Claim 23]

A health functional food for the prevention and improvement of rheumatoid arthritis comprising the Platycodon grandiflorum extract prepared by the method of claim 17 or claim 18 as an active ingredient.

[Claim 24]

An enteric coated oral formulation comprising the Platycodon grandiflorum extr ct prepared by the method of claim 17 or claim 18 as an active ingredient.

[Claim 25]

A method for preventing and treating rheumatoid arthritis containing the step of administering a pharmaceutically effective dose of the Platycodon grandiflorum extract prepared by the method of claim 17 or claim 18 to a subject.

[Claim 26] WO 2016/153262 _Cb₁l . PCT/KR2016/002906

A use of the Platycodon grandiflorum extract prepared by the method of claim 17 or claim 18 for the composition for the prevention and treatment of rheumatoid arthritis. [Claim 27]

A use of the Platycodon grandiflorum extract prepared by the method of claim 17 or claim 18 for the health functional food for the prevention and improvement of rheumatoid arthritis.