Composition for cartilage regeneration comprising isoalantolactone

Abstract

The present application relates to a composition for cartilage regeneration comprising isoalantolactone or a pharmaceutically acceptable salt thereof.

Description

Composition for cartilage regeneration containing isoalantolactone

[0001] The present invention relates to a composition for cartilage regeneration comprising isoalantolactone, and more particularly, to a composition for cartilage regeneration comprising isoalantolactone or a pharmaceutically acceptable salt thereof.

[0002] Cartilage, which protects joints, can be damaged by aging, obesity, diabetes, trauma, and excessive cartilage use. Chondrocytes exist within cartilage, and these chondrocytes play a crucial role in maintaining the balance of catabolism and anabolism, regulating the turnover of type II collagen and aggrecan, which are components of the extracellular matrix (ECM) within cartilage. However, if this balance between catabolism and anabolism within chondrocytes is disrupted by various factors, such as mechanical stress, imbalanced signaling pathways, and cellular changes, the secretion of cytokines (Interleukin-1beta; IL-1β) and the subsequent synthesis and activation of matrix metalloproteinases (MMPs), in particular, can degrade ECM molecules, leading to the gradual destruction of cartilage tissue.

[0003] The above cytokine IL-1β activates various signal pathways, including MAPK (mitogen-activated protein kinase), and the MMPs enzyme activated by the IL-1β promotes the breakdown of type 2 collagen and aggrecan, which are ECM components in cartilage, and this can promote the destruction of cartilage.

[0004] The technology underlying this invention is disclosed in Korean Patent Application Publication No. 10-2004-0107185. The patent discloses a composition containing alantolactone and isoalantolactone that can treat cancer and inflammation, but does not recognize the cartilage regeneration effect of isoalantolactone.

[0005] The present invention aims to solve the problems of the above-mentioned prior art and to provide a composition for cartilage regeneration containing isoalantolactone.

[0006] In addition, the purpose is to provide a pharmaceutical composition for cartilage regeneration or a health functional food composition comprising the composition for cartilage regeneration.

[0007] However, the technical tasks that the embodiments of the present invention seek to achieve are not limited to the technical tasks described above, and other technical tasks may exist.

[0008] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention relates to a composition for cartilage regeneration comprising isoalantolactone or a pharmaceutically acceptable salt thereof as an active ingredient.

[0009] According to one embodiment of the present invention, the isoalantolactone can increase the expression of SoX9 (SRY-Box Transcription Factor 9), but is not limited thereto.

[0010] According to one embodiment of the present invention, the synthesis of type 2 collagen and aggrecan can be promoted by the SoX9, but is not limited thereto.

[0011] According to one embodiment of the present invention, the production of ECM (extracellular matrix) of cartilage is promoted by the type 2 collagen and the aggrecan, thereby allowing cartilage to be regenerated, but is not limited thereto.

[0012] According to one embodiment of the present invention, the expression of MMP3 (Matrix Metalloproteinase 3) and MMP13 (Matrix Metalloproteinase 13) can be inhibited by the isoalantolactone, but is not limited thereto.

[0013] According to one embodiment of the present invention, the MMP3 and the MMP13 can be expressed by cytokines, but are not limited thereto.

[0014] According to one embodiment of the present invention, the cytokine may include, but is not limited to, IL-1β.

[0015] According to one embodiment of the present invention, the MMP3 and the MMP13 can inhibit the regeneration of cartilage by promoting the breakdown of the ECM of cartilage, but are not limited thereto.

[0016] According to one embodiment of the present invention, the composition for cartilage regeneration may additionally include alantolactone, but is not limited thereto.

[0017] In addition, the second aspect of the present invention relates to a pharmaceutical composition for cartilage regeneration, comprising the composition for cartilage regeneration according to the first aspect.

[0018] In addition, the third aspect of the present invention relates to a health functional food composition comprising a composition for cartilage regeneration according to the first aspect.

[0019] The above-described problem-solving methods are merely exemplary and should not be construed as limiting the present invention. In addition to the exemplary embodiments described above, additional embodiments may be included in the drawings and detailed description of the invention.

[0020] According to the above-described means for solving the problem of the present invention, the composition for cartilage regeneration according to the present invention comprises isoalantolactone, and can increase the expression of SoX9 and decrease the expression of MMPs under in vitro and in vivo conditions without toxicity or side effects, thereby promoting cartilage biosynthesis and effectively inhibiting cartilage destruction.

[0021] However, the effects that can be obtained from this center are not limited to the effects described above, and other effects may exist.

[0022] Figures 1a to 1c are graphs analyzing the effect of a composition for cartilage regeneration according to one embodiment of the present invention.

[0023] Figure 2 is a photograph analyzing the effect of a composition for cartilage regeneration according to one embodiment of the present invention.

[0024] Figure 3 is a photograph analyzing the effect of a composition for cartilage regeneration according to one embodiment of the present invention.

[0025] Figure 4 is a graph showing the cytotoxicity of a composition for cartilage regeneration according to one embodiment of the present invention.

[0026] FIG. 5a is a schematic diagram of an experiment to confirm cartilage regeneration of a cartilage regeneration composition according to one embodiment of the present invention, FIG. 5b is a photograph showing the effect of the cartilage regeneration composition on cartilage regeneration, and FIG. 5c is a graph showing the cartilage regeneration effect of the cartilage regeneration composition.

[0027] Figures 6a to 6f illustrate the effect of a composition for cartilage regeneration according to one embodiment of the present invention.

[0028] Figure 7 shows the effect of a composition for cartilage regeneration according to one embodiment of the present invention.

[0029] Figure 8 shows the effect of a composition for cartilage regeneration according to one embodiment of the present invention on a signal transduction pathway.

[0030] Below, with reference to the attached drawings, an embodiment of the present invention is described in detail so that a person having ordinary knowledge in the technical field to which the present invention pertains can easily carry out the present invention.

[0031] However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein. In order to clearly explain the present invention in the drawings, parts irrelevant to the description have been omitted, and similar parts have been designated with similar drawing reference numerals throughout the specification.

[0032] Throughout this specification, when a part is said to be "connected" to another part, this includes not only cases where it is "directly connected" but also cases where it is "electrically connected" with another element in between.

[0033] Throughout this specification, when it is said that a member is located “on,” “above,” “upper,” “lower,” “lower” or “lower” another member, this includes not only cases where the member is in contact with the other member, but also cases where another member exists between the two members.

[0034] Throughout this specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0035] Throughout this specification, the term "type 2 collagen" may be used interchangeably with the terms "type 2 collagen," "collagen II," "type 2 collagen," etc.

[0036] Hereinafter, a composition for cartilage regeneration containing isoalantolactone according to one embodiment and example of the present invention will be described.

[0037] The first aspect of the present invention relates to a composition for cartilage regeneration comprising isoalantolactone or a pharmaceutically acceptable salt thereof as an active ingredient.

[0038] The isoalantolactone according to the present invention is a substance according to the following chemical formula 1, which is derived from the Asteraceae plant, Inula helenium L., and corresponds to a sesquiterpene lactone compound.

[0039] [Chemical Formula 1]

[0040]

[0041] The sesquiterpene lactone compound according to the present invention is found in various plant families such as Asteraceae, Solanaceae, Euphorbiaceae, Lauraceae, and Magnoliaceae, and may include compounds belonging to various types of substances such as isoalantolactone, Artemisinin, Costunolide, Thapsigargin, Arglabin, Parthenolide, Alantolactone, Cynaropicrin, Helenalin, and α-Santonin. The sesquiterpene lactone compound is known to have various pharmacological effects such as anti-tumor, anti-depressant, anti-diabetic, antioxidant, hypoglycemic, vasorelaxant, and hepatoprotective.

[0042] In this regard, the inventors of the present invention confirmed that the isoalantolactone assists the biosynthesis of cartilage and inhibits the destruction of cartilage.

[0043] Specifically, cartilage destruction occurs when the balance between catabolism (destruction of cartilage cells) and anabolism (biogenesis of cartilage cells) within cartilage is disrupted by various factors, including mechanical stress, imbalanced signaling pathways, and cellular changes, leading to the progressive erosion of cartilage tissue. Isoalantolactone can inhibit this cartilage destruction.

[0044] According to one embodiment of the present invention, the isoalantolactone can increase the expression of SoX9 (SRY-Box Transcription Factor 9), but is not limited thereto.

[0045] According to one embodiment of the present invention, the synthesis of type 2 collagen and aggrecan can be promoted by the SoX9, but is not limited thereto.

[0046] According to one embodiment of the present invention, the production of ECM (extracellular matrix) of cartilage is promoted by the type 2 collagen and the aggrecan, thereby allowing cartilage to be regenerated, but is not limited thereto.

[0047] According to one embodiment of the present invention, the expression of MMP3 (Matrix Metalloproteinase 3) and MMP13 (Matrix Metalloproteinase 13) can be inhibited by the isoalantolactone, but is not limited thereto.

[0048] According to one embodiment of the present invention, the expression of MMP3 (Matrix Metalloproteinase 3) and MMP13 (Matrix Metalloproteinase 13) can be suppressed by the SoX9, but is not limited thereto.

[0049] According to one embodiment of the present invention, the MMP3 and the MMP13 can be expressed by cytokines, but are not limited thereto.

[0050] According to one embodiment of the present invention, the cytokine may include, but is not limited to, IL-1β.

[0051] According to one embodiment of the present invention, the MMP3 and the MMP13 can inhibit the regeneration of cartilage by promoting the breakdown of the ECM of cartilage, but are not limited thereto.

[0052] Cartilage destruction primarily occurs through the activation of enzymes, including matrix metalloproteinases (MMPs). These MMPs can promote the breakdown of type II collagen and aggrecan, components of the extracellular matrix (ECM) of cartilage.

[0053] In this regard, the activation of the MMPs enzyme is induced by cytokines including IL-1β. The IL-1β activates various signaling pathways, such as the MAPK pathway, within the chondrocytes. When the MAPK pathway is activated, genes related to anabolic factors are expressed, thereby promoting the production of the MMPs enzyme and inhibiting the expression of Sox9, thereby promoting cartilage destruction.

[0054] The Sox9 (SRY-Box Transcription Factor 9) gene plays an important role in maintaining cartilage synthesis, specifically by promoting the production of extracellular matrix components such as type II collagen and aggrecan. In other words, if the expression of Sox9 is reduced by various factors, the production and repair of the extracellular matrix of chondrocytes may be inhibited, so it is necessary to activate the expression of SoX9.

[0055] Isoalantolactone is a component that increases Sox9 expression. When SoX9 expression is increased, the synthesis of type II collagen and aggrecan is increased, promoting the production of the extracellular matrix of chondrocytes and enabling chondrocyte biosynthesis. In addition, the expression of MMPs enzymes associated with the destruction of chondrocytes, specifically MMP3 and MMP13 enzymes, was suppressed by SoX9 expression.

[0056] Therefore, a composition for cartilage regeneration containing isoalantolactone can assist the biosynthesis of cartilage cells through SoX9 expression and inhibit the destruction of cartilage.

[0057] According to one embodiment of the present invention, the composition for cartilage regeneration may further include, but is not limited to, alantolactone. The alantolactone refers to an isomer of isoalantolactone.

[0058] In addition, the second aspect of the present invention relates to a pharmaceutical composition for cartilage regeneration, comprising the composition for cartilage regeneration according to the first aspect.

[0059] In addition, the third aspect of the present invention relates to a health functional food composition comprising a composition for cartilage regeneration according to the first aspect.

[0060] The pharmaceutical composition for cartilage regeneration according to the second aspect and the health functional food composition according to the third aspect include a cartilage regeneration composition comprising isoalantolactone or a pharmaceutically acceptable salt thereof. Through this, the pharmaceutical composition for cartilage regeneration and the health functional food composition can suppress cartilage destruction and regenerate cartilage.

[0061] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0062] [Example]

[0063] First, 5-day-old ICR mice were killed using CO2 gas, the femur and tibia portions of the knee cartilage were removed, and the cartilage was dissolved by treating with Type 2 collagenase. Then, single chondrocytes were extracted using a 40-mesh screen. Subsequently, the single chondrocytes were 9 x 10 3 DMEM (Dulbecco's Modified Eagle's Medium) containing 10% heat-inactivated FBS and 1% penicillin / streptomycin was added to ensure a cell / well ratio, and 100 μl was dispensed into a 96-well plate. The cells were cultured for 72 hours in an environment of 37°C and 5% CO2 to prepare chondrocytes for confirming the effect of isoalantolactone.

[0064] The chondrocytes used in Experimental Examples 1 to 8 below refer to chondrocytes cultured by a method according to an embodiment, unless otherwise specified.

[0065] [Experimental Example 1]

[0066] Figures 1a to 1c are graphs analyzing the effects of a composition for cartilage regeneration according to one embodiment of the present invention. Specifically, Figures 1a to 1c are for confirming the expression of Type 2 collagen, Aggrecan, and Sox9 by isoalantolactone in vitro.

[0067] Sox9 (SRY-Box Transcription Factor 9) is a transcription factor that plays an important role in the development and maintenance of cartilage, and is a gene involved in the synthesis of cartilage ECM, which includes Type 2 collagen and aggrecan (proteoglycan) and provides structural support and cushioning to joints. In other words, decreased expression and dysfunction of Sox9 promote cartilage synthesis disorder and cartilage destruction. Therefore, to confirm whether isoalantolactone can sufficiently express Sox9 and induce the synthesis of Collagen Type II Alpha 1 Chain (Col2a1) and Aggrecan (Acan), chondrocytes were simultaneously treated with IL-1β and isoalantolactone for 24 hours. In this case, Experimental Example 1 was performed under in vitro conditions.

[0068] Referring to FIGS. 1A to 1C, it can be confirmed that when chondrocytes are treated with IL-1β, the expression of Sox9, Col2a1, and Acan decreases, but when chondrocytes are treated with isoalantolactone, the expression of Sox9, Col2a1, and Acan, which was decreased by IL-1β, increases in a concentration-dependent manner of isoalantolactone.

[0069] [Experimental Example 2]

[0070] Figure 2 is a photograph analyzing the effect of a composition for cartilage regeneration according to one embodiment of the present invention. Specifically, chondrocytes were co-treated with IL-1β and isoalantolactone for 24 hours, and then fixed in a 4% paraformaldehyde solution and embedded in paraffin for histological staining analysis. Subsequently, the tissue was cut into transverse sections using a microtome and analyzed using the Alcian blue staining method. In this case, Experimental Example 2 was performed under ex vivo conditions.

[0071] Referring to Fig. 2, it can be confirmed that the ECM components of cartilage are reduced by IL-1β treatment, but are restored by isoalantolactone. In addition, when chondrocytes were analyzed using the immunohistochemistry (IHC) method, it was confirmed that the protein expression of Sox9, Type 2 collagen, and Acan was reduced in the group treated with IL-1β, but when the group was treated again with isoalantolactone, the protein expression of Col2a1, Acan, and Sox9 was increased.

[0072] [Experimental Example 3]

[0073] Figure 3 is a photograph analyzing the effect of a composition for cartilage regeneration according to one embodiment of the present invention.

[0074] First, a mouse model was created by resecting the meniscus of 10-week-old male C57BL / 6 mice through destabilization of the medial meniscus (DMM). Subsequently, isoalantolactone was injected intra-articularly twice a week for a total of 6 weeks, from 4 to 10 weeks after the meniscectomy surgery. Then, for histological analysis, the knee joints of the mice were extracted 10 weeks after the surgery, fixed in 4% paraformaldehyde solution, decalcified for 2 weeks, and embedded in paraffin.

[0075] Next, when the group injected with isoalantolactone and the group not injected were analyzed in vivo using the immunohistochemistry (IHC) method, the protein expression of Sox9 and Type 2 collagen was reduced in the group of mice in which the cartilage between the joints was lost through DMM surgery (the group not injected with isoalantolactone), but in the group injected with isoalantolactone into the joint, the protein expression of Sox9 and Type 2 collagen increased again.

[0076] [Experimental Example 4]

[0077] Figure 4 is a graph showing the cytotoxicity of a composition for cartilage regeneration according to one embodiment of the present invention.

[0078] Chondrocytes were placed in a medium without FBS (Fetal Bovine Serum) and treated with isoalantolactone at concentrations of 2 μM, 5 μM, 10 μM, or 20 μM, and then cultured at 37°C, 5% CO2 for 24 hours. After 24 hours, the cells were washed with PBS, and 100 μl of serum-free media and 10 μl of WST-1 solution were mixed and treated in each well. After culturing for 2 hours in a dark environment under 37°C, 5% CO2 conditions, the absorbance at 450 nm was measured using a multichannel microplate analyzer. Referring to Figure 4, it can be confirmed that no cytotoxicity was observed when isoalantolactone was treated.

[0079] [Experimental Example 5]

[0080] FIG. 5a is a schematic diagram of an experiment to confirm cartilage regeneration of a cartilage regeneration composition according to one embodiment of the present invention, FIG. 5b is a photograph showing the effect of the cartilage regeneration composition on cartilage regeneration, and FIG. 5c is a graph showing the cartilage regeneration effect of the cartilage regeneration composition.

[0081] A mouse model was created by destabilizing the medial meniscus (DMM) of 10-week-old male C57BL / 6 mice. Isoalantolactone was injected intra-articularly twice a week for a total of 6 weeks, from 4 to 10 weeks after the meniscectomy. Ten weeks after surgery, the knee joints of the mice were extracted for histological analysis, fixed in 4% paraformaldehyde solution, decalcified for 2 weeks, and embedded in paraffin.

[0082] Subsequently, the groups injected with isoalantolactone and those not injected were analyzed in vivo using the Safranin O staining method. As a result, in the group of mice not injected with isoalantolactone after DMM surgery, cartilage was destroyed and the Osteoarthritis Research Society International (OARSI) grade was significantly increased. However, in the group of mice injected with isoalantolactone, the cartilage destroyed by DMM surgery was restored, and it was confirmed that this also reduced the OARSI grade.

[0083] The OARSI grade is a measure used to evaluate the severity of arthritis. A higher OARSI grade indicates more severe arthritis, indicating greater cartilage destruction. Referring to Figure 5c, mice administered isoalantolactone showed a lower OARSI grade, indicating that cartilage regeneration had occurred and arthritis had improved.

[0084] [Experimental Example 6]

[0085] Figures 6a to 6f illustrate the effect of a composition for cartilage regeneration according to one embodiment of the present invention.

[0086] When chondrocytes were treated with IL-1β under in vitro conditions, mRNA expression (Figs. 6a to 6c) and protein expression (Figs. 6d to 6f) of MMP3 and MMP13 increased. Subsequently, when the chondrocytes were treated with isoalantolactone, the mRNA expression (Figs. 6a to 6c) and protein expression (Figs. 6d to 6f) increased by IL-1β decreased in a concentration-dependent manner of isoalantolactone, confirming that isoalantolactone can reduce the expression of mRNA and protein increased by IL-1β.

[0087] [Experimental Example 7]

[0088] Figure 7 illustrates the effect of a composition for cartilage regeneration according to one embodiment of the present invention. Specifically, Figure 7 shows an experiment conducted under ex vivo culture conditions.

[0089] In the group treated with IL-1β in the above chondrocytes, the protein expression of MMP3 and MMP13 increased, indicating that the destruction of chondrocytes was promoted. However, when the group treated with IL-1β was treated with isoalantolactone, it was confirmed that the protein expression of MMP3 and MMP13, which was increased by IL-1β, was reduced again.

[0090] [Experimental Example 8]

[0091] Figure 8 shows the effect of a composition for cartilage regeneration according to one embodiment of the present invention on a signal transduction pathway.

[0092] Typically, treatment of chondrocytes with IL-1β activates mitogen-activated protein kinase (MAPK), which in turn activates several other signaling pathways. In particular, MAPK activation can further contribute to cartilage destruction by inducing the expression of genes associated with catabolic factors and promoting the production of matrix metalloproteinases (MMPs).

[0093] The phosphorylation of three MAPK component proteins (p38, ERK, JNK) was confirmed by pre-treating the above chondrocytes with isoalantolactone for 24 hours and then treating them with IL-1β for 15 minutes. Referring to Fig. 8, it was confirmed that when the above chondrocytes were treated with IL-1β, the phosphorylation of the three MAPK proteins increased. However, when the IL-1β-treated chondrocytes were treated with isoalantolactone, it was observed that the phosphorylation of JNK among the three MAPK proteins decreased.

[0094] Specifically, JNK protein is activated by phosphorylation, and JNK activation can promote cartilage destruction by increasing the expression of MMPs. Therefore, if the degree of JNK phosphorylation decreases, the activity of JNK is inhibited, which means that the expression of MMPs is inhibited. In addition, isoalantolactone only reduces the phosphorylation of JNK, and does not reduce the phosphorylation of ERK and p38. This means that isoalantolactone can selectively inhibit only the JNK protein without affecting other MAPK proteins, and it can be interpreted that isoalantolactone has an effect with drug specificity for JNK. In other words, isoalantolactone can inhibit the production and expression of MMPs.

[0095] The above description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0096] The scope of the present invention is indicated by the claims described below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. A composition for cartilage regeneration comprising isoalantolactone or a pharmaceutically acceptable salt thereof as an active ingredient.

2. In paragraph 1, The above isoalantolactone is a composition for cartilage regeneration that increases the expression of SoX9 (SRY-Box Transcription Factor 9).

3. In paragraph 2, A composition for cartilage regeneration, wherein the synthesis of type 2 collagen and aggrecan is promoted by the above SoX9.

4. In paragraph 3, A composition for cartilage regeneration, wherein the production of ECM (extracellular matrix) of cartilage is promoted by the above type 2 collagen and the above aggrecan, thereby regenerating cartilage.

5. In paragraph 2, A composition for cartilage regeneration, wherein the expression of MMP3 (Matrix Metalloproteinase 3) and MMP13 (Matrix Metalloproteinase 13) is inhibited by the above isoalantolactone.

6. In paragraph 5, A composition for cartilage regeneration, wherein the above MMP3 and the above MMP13 are expressed by cytokines.

7. In paragraph 6, A composition for cartilage regeneration, wherein the cytokine comprises IL-1β.

8. In paragraph 5, A composition for cartilage regeneration, wherein the above MMP3 and the above MMP13 promote the breakdown of ECM of cartilage and inhibit the regeneration of cartilage.

9. In paragraph 1, A composition for cartilage regeneration, wherein the composition for cartilage regeneration further comprises alantolactone.

10. A pharmaceutical composition for cartilage regeneration, comprising a composition for cartilage regeneration according to any one of claims 1 to 9.

11. A health functional food composition comprising a composition for cartilage regeneration according to any one of claims 1 to 9.

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