KR101388597B1 - Composition for prevention or treatment of osteoarthritis including natural pigment of Lithospermi radix and the method for preparing the same - Google Patents

Abstract

The present invention relates to a composition containing a natural pigment of the roots of a root and a method for producing the same for the prevention and treatment of osteoarthritis.
The composition containing the branch root extract of the present invention shows a significant effect on osteoarthritis even when used alone, it can be widely used as a osteoarthritis preventive and therapeutic agent or osteoarthritis health functional food.

Description

Composition for prevention or treatment of osteoarthritis including osteoarthritis including natural pigment of Lithospermi radix and the method for preparing the same}

The present invention relates to a composition containing a natural pigment of the roots of a root and a method for producing the same for the prevention and treatment of osteoarthritis.

Osteoarthritis is osteoarthritis, which is a degenerative arthritis that is caused by degenerative changes in the cartilage and the surrounding bone in the lubricated joint, and is a different form of arthritis than rheumatoid arthritis.

Osteoarthritis is characterized by a gradual loss of articular cartilage, hypertrophy of bone located below the cartilage, bone formation at the articular edges, and nonspecific synovial inflammation.

Osteoarthritis is the cartilage change stage (step 1), which causes swelling due to increased water content in the cartilage, and the fibrillation step (step 2), in which the cartilage surface is cracked and torn as the cartilage is broken and torn, revealing bones and narrowing the joint cavity. Cartilage cells begin to produce cartilage to repair cartilage, but cartilage destruction occurs faster than cartilage production (step 3), resulting in reduced cartilage overall, bone changes that lead to deformed joints and dysfunction. Step (step 4) and the soft tissue (tissue softening) thickening step (step 5).

On the other hand, rheumatoid arthritis is a chronic autoimmune disease characterized by inflammation and proliferation of synovial cells, and unlike osteoarthritis, osteoporosis and osteoporosis of the periarticular bone occur.

Rheumatoid arthritis is a condition in which inflammation of the synovial membrane is spread to the joint capsule, ligament, tendon (stage 1), gradual destruction of the joint cartilage, (Stage 2) in which the tension of the joints and ligaments disappears (step 3), in which the inflammation penetrates into the bones, resulting in partial erosion of the bones (step 3), and the step of loss of joint function (step 4).

Looking at the difference between rheumatoid arthritis and osteoarthritis, since rheumatoid arthritis is an autoimmune disease, severe inflammation is caused and cartilage destruction is a secondary symptom due to severe edema. Cartilage destruction is a major symptom caused by external factors such as shock, and this is why the swelling, inflammation, and joint deformations are accompanied by secondary symptoms.

Therefore, rheumatoid arthritis and osteoarthritis are generally classified as other diseases, and the treatment methods for treating are different.

On the other hand, Lithospermum erythrorhizon Sieb. Et Zucc. Is a perennial herbaceous herbaceous plant grown in Jecheon and Jindo, Korea. It has been used as a herbal medicine and food coloring.

Licorice is mainly used to promote blood circulation, antipyretic and detoxification, or to treat hemostasis, hematuria, constipation, burns, eczema and urinary tract infections (Ahn et al., 2009; Lee et al., 1998). In addition, it has been reported that the anti-influenza effect in recent years (Tian et al. 2011).

By the way, it has recently been disclosed that the complex extract including baekryechae and gyeji extract in addition to the licorice extract has the effect of preventing and improving rheumatoid arthritis (Korean Patent Publication No. 10-2009-0095740) (hereinafter referred to as '95740 patent').

By the way, the 95740 patent only discloses a licorice extract by a polar solvent such as ethanol, rather, the licorice extract alone has only a weak effect of preventing and treating arthritis, and only a significant effect when mixed with white bran and gyeji extract It is disclosed.

That is, the 95740 patent shows that when the herb extract is used alone, there is almost no effect of preventing and treating arthritis.

In order to solve the problems of the prior art as described above, the present invention is an extraction method that can extract the osteoarthritis pharmacological effect components from the roots of borage so that the root extract alone can have a significant prophylactic and therapeutic effect It is intended to provide a branch root extract extracted by the method.

In addition, the present invention is to provide a composition having a preventive and therapeutic effect of osteoarthritis, including the branch root extract.

Osteoarthritis therapeutic agent according to one feature of the present invention for solving the above problems contains an effective amount of natural pigment of the bored root.

In addition, the osteoarthritis preventive agent according to another feature of the present invention contains a natural pigment of the bored root in an effective amount.

In addition, the health functional food for osteoarthritis improvement and prevention according to another feature of the present invention contains the natural pigment of the bored root.

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According to the branched root extraction method according to the present invention, the natural pigment component useful for osteoarthritis can be easily extracted from the branched root.

Accordingly, the branched root extract according to the present invention can achieve excellent therapeutic and prophylactic effects on osteoarthritis even when used alone.

Chichi root extract of the present invention can be usefully used in pharmaceutical compositions or foods for the prevention and treatment of osteoarthritis.

Figure 1 shows the histopathological arthritis index of the knee cartilage of the supercritical extract of the dentate root of the present invention and the MIA-induced osteoarthritis rats of acetylcyconin derived from the dendritic root.
Figure 2 shows the inhibitory ability of cartilage collagen degrading enzymes in the serum of MIA-induced osteoarthritis rats of acetylcyconin from the supercritical extract of the dendritic root of the present invention.
Figure 3 shows the degree of histopathological damage of the knee cartilage of the supercritical extract of the deciduous root of the present invention and the MIA-induced osteoarthritis of acetylcyconin derived from the deciduous root.

The present inventors have found that the pharmacologically effective component useful for osteoarthritis can be extracted by using a nonpolar solvent or supercritical extraction of the roots of boredom.

In addition, the inventors of the present invention are natural pigment components contained in large amounts in the roots, ie, acetylshikonin (acetylshikonin), shikonin (shikonin), alkannan (alkannan), isobutyl-sikonin (isobutyryl-shikonin), β, Branched extracts containing various siconin derivatives such as β-dimethyl-acrylshikonin, β-hydroxy isovaloryl shikonin, and teracryl shikonin It was first identified that it exhibits useful pharmacological effects in osteoarthritis animal models.

In addition, the present inventors confirmed that the siconin derivative, which is a pharmacologically effective component and a natural pigment component, was not extracted by polar solvent extraction such as ethanol and alcohol, which are conventional extraction methods. Therefore, it was found that the polar solvent extracts of the roots (eg, ethanol extracts, etc.) showed no therapeutic and prophylactic effects on osteoarthritis.

In addition, the present inventors confirmed that acetylsiconin among the sikonin derivatives contained in the branch root extract has an inhibitory effect on the expression of MMP-1 (collagenase-1), a collagen degrading enzyme involved in the destruction of cartilage tissue. However, osteoarthritis is caused by the destruction of cartilage tissue, and acetylsiconin, which is a major component of the borage root extract, inhibits the expression of collagenase, thereby demonstrating the useful therapeutic and prophylactic effect of the borage root extract in osteoarthritis.

Therefore, the branched root extraction method of the present invention includes a method of extracting the branched roots without using a polar solvent such as ethanol, supercritical extraction or using a nonpolar solvent.

Here, the nonpolar solvent includes hexane, ethyl acetate and the like, and hexane is particularly preferably used.

The branched root extract extracted by the branched root extracting method of the present invention is expected to be an excellent effect as a therapeutic or preventive agent for osteoarthritis.

In addition, the branched root extract extracted by the branched root extract method of the present invention can be usefully used for health functional foods to prevent and improve osteoarthritis.

Hereinafter, the present invention will be described in detail with reference to the preferred embodiments, which will be readily apparent to those skilled in the art to which the present invention pertains. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Example  One. Shag  Root Supercritical  extract

4300 g of branch root samples were extracted according to the supercritical extraction conditions of Table 1 below, to obtain 47.35 g of supercritical extract, wherein the yield of the final extract was 1.1%.

Here, Chichi root samples were purchased from Korea Herbal Medicine Association for 1 year, and the voucher specimen (MPS000071) is stored in the plant specimen room of the ginseng specialty department of the National Horticulture & Special Research Institute of Rural Development.

Item Supercritical Extraction Conditions Device used 10L extractor Extraction pressure 400 bar Extraction temperature 60 ° C Extraction time 5 hours Sample amount 4300 g Extraction amount 47.35 g Extraction yield 1.10%

Example  2. Shag  Root Hexane  extract

Put 150 mL of hexane into 150 g of branched root sample, ultrasonically extract for 30 minutes, filter, and then add the same amount of hexane to the residue, extract the mixture 10 times in the same way, filter, and concentrate the extract with a vacuum rotary concentrator and then freeze-dryer. Drying was carried out to remove the solvent completely to obtain a final 1.8g of hexane extract, the extraction yield was 1.2%.

Comparative Example  One. Shag  Root Ethanol Alcohol Extract

Put 150 mL of ethanol alcohol into 150 g of branch root samples, and ultrasonically extract for 30 minutes, filter, and then add the same amount of ethanol alcohol to the residue, extract the mixture 10 times in the same way, filter, and concentrate the extract with a vacuum rotary concentrator and then again. Drying with a lyophilizer to completely remove the solvent to obtain a final 36.456g of ethanol alcohol extract, the extraction yield was 17.8%.

Test Example  One. Acetyl Siconin  Content analysis

2 mL of methanol was dissolved in 2 mg of the branch root extracts of Examples 1 and 2 and Comparative Example 1, respectively, and the resultant was filtered through a membrane filter of 0.45 μm and used as a sample for HPLC analysis. HPLC analysis conditions were as shown in Table 2 (acetylsyconin content HPLC analysis conditions of the branch extract). At this time, the acetylsiconin standard for HPLC was purchased from Nakalai tesque, Inc., Japan. Acetonitrile was used as HPLC grade solvent (J. T Baker ^® , Nederland), and GR grade solvent was used for extraction of borage root samples. HPLC was used with an Agilent 1100 series HPLC system (Agilent Technolgies, USA).

HPLC column: Zorbax Eclipse XDB-C18 (4.6x150mm, 5㎛)
2. Column temperature: 25 ℃
3. Detector wavelength: UV 520nm
4. Flow rate: 0.8ml / min
5. Mobile phase Time (minutes) % (Ultra pure water) (Acetonitrile) 0 45 55 17 20 80 17.1 0 100 22 0 100 22.1 45 55 25 45 55

As a result, from Table 3 (acetylaceconin content according to the extraction method of the branch), in Examples 1 and 2, the acetylsiconin content per mg mg extract was analyzed as high content as 210, 203μg / mg, Comparative Example It can be seen that 1 does not detect acetylcyconin. From this, it is presumed that the branched siconin pigment component is a nonpolar substance as a naphthoquinone component, so it is well eluted by hexane or supercritical extraction but is not well eluted with a polar solvent such as ethanol alcohol. .

Active ingredient Ethanol alcohol extract
(Comparative Example 1) Hexane extract
(Example 2) Supercritical Extract (CO ₂ Extraction)
(Example 1) Acetyl Siconin
(μg / mg) - 203 ± 19 210 ± 10

Example  3. Exhaustive  Active ingredient Acetylcyconin  Separation tablet

12 L of hexane was added to 3 kg of powder samples of branched roots, and the mixture was left at room temperature for 10 days, extracted four times in the same manner, filtered and concentrated in vacuo. 6 L of the hexane solvent was added to the remaining sample residue, followed by extraction with shaking at room temperature, followed by filtration and concentration in vacuo.

The concentrated hexane concentrate (Hexane ext., 26 g) was subjected to column chromatography on a silica gel packed column (5 × 40 cm).

For elution solvents, 500 mL fractions were eluted by gradient elution of Hexane: EtOAc (30: 1 → 1: 11) and EtOAc: MeOH (25: 1 → 1: 2) mixed solvents. It was. Each preparative fraction was identified by HPLC analysis at a wavelength of 520 nm UV detector, and then similar fractions were combined and reconcentrated to obtain 30 final concentrated fractions (Fr. 1-30). Fr. in the last 30 fractions. 6, 7 fractions were precipitated with MeOH to finally separate and purify compound 1 (1.1 g). JEOL JNM-LA400 FT-NMR spectrometer was used for the NMR measurement of spectroscopic analysis to identify the structure of the isolated compound. NMR and mass spectrometry confirmed that this compound was acetylcyconin.

Test Example  2. MIA Rat  Pharmacological effects on arthritis

Male Sprague-Dawley rats (Orient Bio, Korea) with a body weight of about 160 g were purchased and used in the experiment after being adapted for 1 week in a clean animal breeding room (temperature 23 ± 1, humidity 55 ± 5%).

Feed and water were freely consumed during the experiment.

Osteoarthritis was induced by a modification of the method of Guzman (Toxicologic Pathology, 31: 619-624, 2003). MIA solution was prepared by dissolving monosodium iodoacetate (hereinafter referred to as “MIA”) (Sigma, USA) at 5 mg / ml in physiological saline for injection.

0.5% carboxymethyl cellulose was added to 200 mg / kg as a negative control (indicated as 'G1' in FIG. 1), and a green leaf known as osteoarthritis effect as a positive control (indicated as 'G2' in FIG. 1). Mussel oil extract is added to 150mg / kg, supercritical extract obtained in Example 1 (indicated by 'G3' in Figure 1) is added to 200mg / kg, Example 3 ('G4' in Figure 1) Acetylsiconin compound obtained in the above) was added to 5 mg / kg.

At the beginning of the experiment, Zoletile: Rumpun (2: 1) was anesthetized by intramuscular injection, and the anterior sides of both knee joints were disinfected. Then, MIA solution (1mg / 50 ul) was injected into the knee joint with a hemilton syringe (26 guage) in the middle of the knee cartilage. The drug (negative control, positive control, Example 1 and Example 3) was injected by precise injection into the site (between cartilage). At this time, the normal group was injected with 50 ul of saline for injection in each of the two knee joints.

Thereafter, the body weight was measured once a week, and the blood was collected at day D22, and the serum was separated and stored at -70 ° C. The knee cartilage of each individual with MIA induced osteoarthritis was separated and fixed and demineralized. After staining with & E (Hematoxyline & Eosin), articular cartilage tissue was observed under a microscope.

Histopathological items include subchondral bone change, chondrocyte necrosis, cartilage erosion, osteophyte and cartilage cleft, with a score of 0-5 points per item. It was performed by a toxic pathologist.

Speculum scores are expressed as mean and standard deviation, and the results were tested using the nonparametric Kruskal-Wallis test. Kruskal-Wallis test was used to determine the significance level of p <0.05. Statistical analysis was performed using Dunn's multiple comparison test. Statistical analysis of each group was performed using GraphPad PRISM ^® Version 4.0 (GraphPad Software, USA).

As a result, as can be seen in Figure 1, compared to the negative control group (G1) and positive control group (G2), the arthritis index improvement effect is statistically significant in the branch treatment root extract treatment groups of Examples 1 and 3 of the present invention. You can see that it appeared. On the other hand, the positive control group (G2) supplemented with green lipped mussel oil extract, which is known as a functional ingredient for improving joint health, did not improve the arthritis index.

In addition, as can be seen in Figure 2, compared to the negative control group (G1), in the Chichi root extract treatment group of Examples 1 and 3 statistically significant inhibit the expression of cartilage collagen degrading enzyme (MMP1) content The reducing effect was confirmed. From this, it can be seen that the branch root extract of the present invention inhibits the expression of cartilage collagen degrading enzyme (MMP1) that causes osteoarthritis.

In addition, as can be seen in Figure 3, in the negative control group (B) it was observed that osteoarthritis is induced by MIA, cartilage tissue is severely damaged and destroyed.

On the other hand, the Chichi root extract treatment group (E) of Example 1 showed the cartilage tissue state similar to that of the normal group (A), which was evaluated because the chichi root extract inhibited the enzymatic activity of MMP1 and inhibited cartilage destruction. .

On the other hand, damage to the cartilage tissue was also recovered in the acetylsiconin treated group (D) of Example 3, and it was observed that the cartilage tissue was better than the negative control group.

From the above results, the supercritical extract and the acetylsiconin component of the roots of the branched roots of Examples 1 and 3 are excellent in inhibiting cartilage destruction and improving osteoarthritis.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Do.

Claims (16)

A therapeutic agent for osteoarthritis, which contains an effective amount of acetylcyconin, a natural pigment of borage roots, and inhibits MMP-1 activity, a collagen degrading enzyme. delete delete An osteoarthritis preventive agent containing an effective amount of acetylcyconin, a natural pigment of the branch root, and inhibiting MMP-1 activity, a collagen degrading enzyme. delete delete Comprising a natural pigment of the bored root acetylsikonin, a composition for health functional food for the prevention and improvement of osteoarthritis. delete delete delete delete delete delete delete delete delete

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