KR100407557B1 - Hard tissue regeneration accelerator composition comprising Eochocho extract - Google Patents

Abstract

The present invention relates to a hard tissue regeneration accelerator composition comprising Echochocho extract.

The therapeutic agent of the present invention promotes proliferation of osteoblasts and periodontal ligament cells and increases alkaline phosphatase activity of the cells, thereby promoting hard tissue regeneration and proliferation, as well as a form capable of functioning as hard tissue, that is, sufficient hardness. Eochochocho extract which can form the hard tissue which has is included.

Therefore, the therapeutic agent of the present invention is a natural-derived drug containing an extract obtained from Eoseongcho, which is one of the herbal medicines, and because it has excellent side effect of regenerating and proliferating hard tissue without showing side effects, diseases related to hard tissue regeneration and proliferation, such as osteoporosis and periodontal disease, etc. It provides a hard tissue regeneration accelerator composition that can be used safely in place of existing therapeutic agents for the purpose of prevention and treatment of.

Description

Hard tissue regeneration accelerator composition comprising Eochocho extract

The present invention relates to a hard tissue regeneration accelerator comprising Echochocho extract.

Hard tissues present in the human body are largely classified into bones and teeth. Representative diseases caused by problems with such hard tissues include osteoporosis and periodontal disease.

Osteoporosis, also known as osteoporosis, is a condition in which the total bone mass decreases and a hole is formed in the bone, which causes the bone to be easily broken or bent by a weak impact. Osteoporosis is commonly found in the elderly and is the cause of fractures such as femur fractures and vertebral fractures. In the United States, 1.3 million cases of osteoporosis fractures occurred in 1985, resulting in $ 7 billion in medical expenses. The disease is caused by menopause, aging, hyperfunction of the thyroid or parathyroid gland, chronic renal failure and the administration of corticosteroids. The most common is a decrease in estrogen after menopause in women.

Bone tissue is composed of two-thirds of the inorganic material, one third of the organic material, the former is mainly calcium phosphate (Calcium Phosphate), the latter is mainly collagen (collagen). Osteoblasts, osteoclasts, and osteocytes are involved in bone formation and resorption in bone tissue. Osteoblasts play a decisive role in regenerating double bone. Osteoblasts are located on the surface of the bone to be produced, and synthetic collagen fibers (collagen fibers) required for bone formation. In addition, neutral and alkaline phosphatase is widely distributed in the cell membranes of osteoblasts, and PO ₄ ^-3 / Ca ⁺² is deposited by these to cause calcification of bone. That is, bone regeneration is in progress. This fact means that osteoblasts do not induce osteoporosis because bone regeneration progresses rapidly when the number of osteoblasts increases, and osteoporosis can be treated by increasing the number of osteoblasts. Rat calvarial cells of the white paper used in the experimental example of the present invention are osteoblasts isolated from the skull of rats and are generally used for bone formation research.

In fact, the mechanism of action of many substances that promote bone formation is known to induce proliferation of osteoblasts. For example, growth hormone (GH), like insulin growth factor I (IGF-I), and transforming growth factor beta (TGF-beta) are known to be osteoblasts. It is a growth factor that promotes bone growth by promoting the growth of. Estrogen, which is effective in treating osteoporosis, is also known to promote osteoblast proliferation.

On the other hand, in alcoholic patients, bone breaks easily because alcohol inhibits the proliferation of osteoblasts, and bone-related diseases in the elderly are at least partially caused by decreased proliferation of osteoblasts. Glucocorticoid hormone is a substance administered during the treatment of asthma, which also suppresses the proliferation of osteoblasts and causes osteoporosis.

Drugs currently being used in the treatment of osteoporosis include estrogen, ipriflavone (ie, 7-isopropoxy isoflavone), bisphosphonate, vitamin K ₂ , calcium preparations, vitamin D, and calcitonin. Estrogens, bisphosphonates, calcitonins, and vitamin K ₂ inhibit bone breakdown, and calcium preparations and vitamin D have been shown to treat osteoporosis by increasing blood calcium levels. Ipriflavones, which have been recently isolated from natural products, are synthetic derivatives of isoflavone, a major component of soybeans, which promote osteoblast proliferation and differentiation and increase collagen synthesis and calcified nodules.

These drugs have a number of side effects. First, estrogen preparations are characterized by rashes, irregular bleeding, lower abdominal pain, jaundice, nausea, vomiting, general malaise, weight gain, and contraindications for ovarian tumors. Bisphosphonates (eg, Palmidronate) are the most common side effects of fever and cause malaise, thrombophlebitis at the site of administration, and hypophosphatemia in patients with Paget's disease. In addition, osteoporosis appears at the beginning of administration, and dose-dependent nausea and abdominal pain occur during oral administration. In addition, transient leukopenia occurs. Calcitonin (including Elcatonin and Salcatonin) generally causes shock, etc., so it is necessary to make sure that it is sufficiently questioned prior to administration and to conduct an intradermal reaction in advance. Hypersensitivity reactions may also occur, including hot flashes, hot flashes, chest tightness, nausea, vomiting, anorexia, diarrhea, dry mouth, dizziness, hyponatremia, pruritus, asthma attacks, frequent urination, and edema. A side effect of vitamin K ₂ is stomach discomfort. Epriflavones are the most common side effect of abdominal discomfort, such as loss of appetite, nausea, vomiting, diarrhea, rash, pruritus.

Therefore, there is an urgent need for the development of drugs derived from natural products, which are thought to have relatively low onset of side effects in order to compensate for such disadvantages of existing drugs.

Periodontal disease is a disease in which gingival tissue and alveolar bone are lost due to bacterial inflammation, resulting in tooth extraction. Stepwise treatment is used according to the progression. The principle of treatment is to remove the cause, delete soft and hard tissues affected by the disease, and promote tissue regeneration. Ideal healing of periodontal tissue requires periodontal ligament reconstruction connecting teeth and alveolar bone and requires surgical procedure. The most important thing in this healing process is the proliferation of osteoblasts, followed by the attachment of the periodontal ligament cells to the tooth surface. Periodontal ligament cells have biochemical properties similar to osteoblasts and are multifunctional cells that differentiate into cementum, a bone tissue covering osteoblasts and roots. In other words, periodontal ligament cells are differentiated to form hard tissue. Therefore, the surgical procedure for the improvement of the microenvironment that can affect the cell remodeling of the periodontal ligament is important, but the development of a method for increasing the proliferation capacity and activity of the periodontal ligament cells and osteoblasts is important. In recent years, the development of new drugs has been actively attempted.

First, the effect of several polypeptide growth factors (polypeptide growth factor) has been demonstrated in vitro (in vitro), but there is a problem in clinical application because the problem of stability has not yet been solved. In addition, the extraction process is complicated, takes a long time and is expensive, the clinical application is impossible. To date, the only natural-derived drug developed as a periodontal treatment at home and abroad is corn (Zea Mays L.) extract and is marketed under the trade name Insadol. The mechanism is not known and is only applied based on clinical data. In Japan, the extracts of Geumgeunhwa and Yeongyo have been used for the treatment of periodontal disease by topical drug delivery and have reported the effect on gingival fibroblasts on in vitro. Thimble root (Scutelariae Radix) has been reported to have anti-inflammatory and antipyretic effects, and the anti-inflammatory action of quercitrin has been reported, and the pharmacological action of natural products continues.

Recently, Scutelariae Radix and Centella asiatica have been reported to increase the activity of epithelial proliferation and periodontal ligament cells, and red ginseng saponin has been studied in the growth and differentiation of cultured periodontal ligament cells. It has been reported to increase the proliferation and calcification nodule formation and increase the activity of alkaline phosphatase. The control (Zizyphus Fructus) has a significant chemotactic effect on gingival fibroblasts and periodontal ligament cells. Magnolol and hinokiol have also been shown to have inhibitory effects on cytokine production, an inflammatory mediator.

However, there is no current development as a drug exhibiting an excellent effect on periodontal disease, and more recently, there is an increasing interest in the development of drugs that can prevent and treat diseases without harm to the human body at home and abroad. In order to treat periodontal disease, the researches that have been studied so far are limited to substances that exhibit inflammatory healing and suppression of inflammatory response, periodontal ligament cell growth, or gingival cell proliferation, and to treat periodontal disease by increasing osteoblast proliferation and activity. There was no attempt.

The present inventors have made a lot of research and efforts on substances showing osteoblast proliferation and differentiation effect among various natural extracts to develop drugs that can be applied to hard tissue-related diseases such as osteoporosis and periodontal disease by showing osteoblast proliferation and differentiation effect. As a result, the extract obtained from Eoseongcho was found to have osteoblast proliferation and differentiation effect, thereby completing the present invention.

Houttuniae Herba, also known as succulents, is obtained by harvesting and drying ground portions of Houttuynia cordata THYMB. In addition to the 28 essential oils, there are reported quercetin, isoquencetin, rutin, hyperin and afserin. It is known to show anti-inflammatory and antimicrobial effects (Yuk Chang-soo et al., 12 persons, modern herbal medicine, Seoul, Hakchangsa, 24-128, 1993).

An object of the present invention is to provide a hard tissue regeneration accelerator composition comprising Echochocho extract to treat diseases related to hard tissue regeneration and proliferation such as osteoporosis, periodontal disease and the like.

The present invention relates to a hard tissue regeneration accelerator composition comprising Echochocho extract.

Fish extract of the present invention can be extracted according to the following herbal extract method:

The fish paste is added to the extraction solvent and warmed at 50 to 120 ° C. for 5 to 24 hours. The extract is cooled to 40-60 ° C. and filtered to obtain a supernatant. This extraction and filtration is repeated one or more times to combine the supernatant, and the solvent is concentrated by evaporation. Preferably, the method of warming heating at 80-100 degreeC in a water bath is used.

Alternatively, fishery vinegar is added to an extraction solvent, cooled at room temperature or 4 ° C. for 5 to 7 days, and filtered to obtain a supernatant. This extraction and filtration is repeated one or more times to combine the supernatant, and the solvent is concentrated by evaporation.

In the two extraction methods as described above, it is preferable to grind fishery vinegar in a small size, and a preferred example of the extraction solvent is 50-100% aqueous methanol solution. The extraction and filtration operations are preferably repeated three times, and the concentrate may be dried to obtain a powder.

Hard tissue regeneration accelerator composition according to the present invention by promoting the proliferation of osteoblasts and periodontal ligament cells and increase the alkaline phosphatase activity of the cells to exhibit a hard tissue regeneration and proliferation effect as well as a form that can function as hard tissue Echo herb extract capable of forming hard tissue with sufficient hardness, and thus can be used as a direct or adjuvant treatment for various diseases associated with the phenomenon of hard tissue regeneration or reduced hard tissue proliferation. Applicable diseases include osteoporosis and periodontal disease. In addition, it can be used for the purpose of preventing periodontal disease by adding the therapeutic agent of the present invention to a toothpaste additive or a gargle solution and can also be used for the purpose of strengthening egg shells.

Hard tissue regeneration accelerator composition according to the present invention may be composed only of the extract of Echochocho may also further comprise other active ingredients and pharmaceutical additives. For example, when the therapeutic agent of the present invention is used in periodontal disease, it is possible to obtain a more excellent effect by including the drug with an anti-inflammatory effect.

Hard tissue regeneration accelerator composition according to the present invention is administered in a solid, semi-solid or liquid form of pharmaceutical formulation suitable for oral or parenteral administration by combining a pharmaceutically acceptable inert carrier and Echochocho extract during clinical administration can do. Pharmaceutically acceptable inert carriers which may be suitably used for this purpose may be solid or liquid and any one or more of a substance which can act as a diluent, flavor, solubilizer, lubricant, suspending agent, binder, disintegrant Can be.

Hard tissue regeneration accelerator composition comprising the Echochocho extract of the present invention for use in the treatment of hard tissue regeneration or proliferation-related diseases is preferably a dosage of 0.01 ~ 0.10 g of Echochocho extract as a dry powder per kg of body weight per day. However, dosage may vary depending on the needs of the patient, the extent of the condition to be treated, and the compound to be used.

Hereinafter, the present invention will be described with reference to specific examples, but the present invention is not limited thereto. However,% in this specification and an Example means the volume%.

Example 1

100 g of eosungcho was added to 500 ml of 80% methanol (methanol: water = 4: 1), equipped with a reflux condenser, and warmed for 12 hours in a 95-100 ° C. water bath. The extract was cooled to about 50 ° C. and filtered through several layers of gauze to obtain a supernatant. This extraction and filtration was repeated three times to combine the supernatants and concentrated by evaporating methanol completely under reduced pressure using a rotary evaporator. It was dissolved in a small amount of distilled water.

Example 2

The Echochocho solution finally obtained in Example 1 was frozen at -80 ° C and lyophilized to obtain a powder.

Example 3

100 g of eosungcho was ground to a small size and placed in 500 ml of 80% methanol (methanol: water = 4: 1). This extraction and filtration was repeated three times to combine the supernatants and concentrated by evaporating methanol completely under reduced pressure using a rotary evaporator. It was dissolved in a small amount of distilled water.

Example 4

The Echochocho solution finally obtained in Example 3 was frozen at -80 ° C and lyophilized to obtain a powder.

Preparation Example 1 Preparation of Tablet

5.0 mg fish extract of Example 2

Lactose BP 150.0 mg

Starch BP 30.0 mg

Pregelatinized Corn Starch BP 15.0 mg

1.0 mg magnesium stearate

Echochocho extract of Example 2 was sieved and mixed with lactose, starch and pregelatinized corn starch, then a suitable volume of purified water was added and granulated into a powder. The granules were dried and then mixed with magnesium stearate and compressed to obtain tablets.

Preparation Example 2 Preparation of Capsule

5.0 mg fish extract of Example 2

Starch 1500 100.0 mg

Magnesium Stearate BP 1.0 mg

Echochocho extract of Example 2 was sieved, mixed with excipients, and filled into gelatin capsules to obtain capsules.

Preparation Example 3 Preparation of Syrup

5.0 g of Eochocho extract of Example 2

637.5 g per bag

2.0 g of sodium carboxymethylcellulose

0.28 g of methyl paraben

0.12 g of propylparaben

20 ml of ethanol

First, white sugar was dissolved in 500 ml of purified water. Sodium carboxymethylcellulose was separately dissolved in 400 ml of purified water and mixed with the solution. Methyl paraben and propyl paraben were added and dissolved therein, followed by ethanol and purified water to make 1000 ml. The Echochocho extract of Example 2 sieved here was suspended, and the syrup agent was obtained.

Preparation Example 4 Preparation of Pasta (Toothpaste)

50.0 g of Eochocho extract of Example 2

50.0 g of corn starch

100.0 g of white petrolatum

First, the eochochocho extract and corn starch of Example 2 were sieved and added to white petrolatum to soften the whole starch until homogenous to obtain a pasta agent.

Preparation Example 5 Preparation of Gargle Solution

10.0 g of eochocho extract of Example 1 (as weight of dry powder)

Mint 50.0 ml

Purified water added 1000.0 ml

First, Echochocho extract of Example 1 was dissolved in purified water, peppermint water and purified water were added to this, and it made the whole quantity, and it filtered and obtained the gargle solution.

Experimental Example 1: Effect of Proliferation of Rat Cranial Canal Cells

In order to confirm the effect of Echochocho extract obtained in Example 1 on the proliferation rate of osteoblasts, the following experiment was performed using rat calvaria cells of white paper.

First, two dog cells of white paper were cultured by the following method. After anesthesia, pentobarbital sodium (Tokyo Industrial Chem., Japan) was injected into the white paper before and after the weight of 100 g, the scalp was washed with 70% alcohol solution to disinfect, and the head and neck were dropped and sacrificed. Surgical scissors were used to separate the mandible from the maxilla and peel the scalp. Isolate the exposed cranial canal, completely remove the soft tissue, and then cut and cut into 200 U / ml penicillin (Gibco, USA) and 200 μg / ml of streptomycin (Gibco, USA). It was washed five times in Minimum Essential Cultures (Dullbeco's Minimum Essential Medium, DMEM) (Gibco, USA). This was again sliced and then evenly distributed in a 35 mm cell culture dish. To this was added DMEM containing 20% Fetal Bovine Serum (FBS), 100 U / ml penicillin and 100 μg / ml streptomycin, which was the initial culture medium, and 5% CO at 37 ° C. and 100% humidity. ₂ cultured in an air mixed incubator (Vision, Korea). After 2 generations, the culture medium was replaced with DMEM containing subculture 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 3 days intervals, until they were grown.

0.5 ml of the culture solution (containing 1 × 10 ⁴ white cranial canal cells) was placed in a 24-well culture dish (Corning Co., USA) and incubated for 24 hours in an incubator. After confirming that the cells completely attached to the bottom of the culture dish, the supernatant was removed. The cell culture dish was divided into one control group and the experimental group, and the control group was not injected with the drug, and the experimental group was injected with each culture dish so that the final concentration was 1 μg / ml. Control and experimental groups were cultured using subculture. Discard the broth in each dish on day 2 and 14 of the culture and wash with phosphate-buffered saline (PBS, phosphate buferred saline) and 0.05% trypsin / 0.02% editai (EDTA) (Gibco, USA Cells were completely separated from the culture dish, stained with trypan blue, and the number of cells was measured using a hemocytometer on an inverted microscope (Olympus Co. Japan). The results were as shown in Table 1 below.

TABLE 1

As a result of analyzing the results of the experiment compared to the control group not administered the drug, the standard deviation was large at the time of short-term administration (cell culture 2 days), and therefore, no significant difference was found between the experimental data. At 1) it was confirmed that the fish extract obtained in Example 1 significantly increased the proliferation of white cranial canal cells. Therefore, it can be seen that the therapeutic agent containing the fish extract according to the present invention has excellent osteoblast proliferation effect.

Experimental Example 2: Experimental effect of increasing alkaline phosphatase activity using white cranial canal cells

In order to determine the effect of Echoseongcho extract on the activity of alkaline phosphatase in the cranial canal cells of the white paper, the following experiment was conducted.

Cells were harvested from the cranial tubes of the white paper and cultured in the same manner as in Example 1. Trypsin treatment was performed to determine the number of two canine cells in the initial dense state, and then inoculated 1 × 10 ⁵ cells per well into 24 well tissue culture plates (Corning, USA). In the subculture, Eochocho extract was injected into the medium so that the final concentration was 1 μg / ml, and the culture solution was discarded on the 2nd and 14th days of culture, washed three times with phosphate buffered saline solution, and then in the cell layer. 1 ml of lysis buffer (0.02% Nonident P-40) was added and ground for 30 seconds using an ultrasonic pulverizer (Ultrasonic Dismembrator Model-300, Fisher, USA), and 300 µg was taken to measure the activity of alkaline phosphatase. .

The measurement of alkaline phosphatase activity was performed as follows with reference to the method of Bessay et al. And the method of Burch et al. As a standard solution, paranitrophenol was added and reacted at 37 ° C. for 30 minutes, and then 1N NaOH was added to stop the reaction. The absorbance was measured with a spectrophotometer (Shimatsu, Japan) at 410 nm, and the activity value of alkaline phosphatase was shown using the unit as nmol / 30min / mg protein, ie, IU.

TABLE 2

As a result of the experiment, it was confirmed that the Echochocho extract obtained in Example 1 significantly increased the activity of alkaline phosphatase when compared with the control group not administered the drug. Alkaline phosphatase is an enzyme that plays a crucial role in the formation of hard tissues, ie calcification of hard tissues by depositing PO ₄ ^-3 / Ca ⁺² on the cell membrane of osteoblasts. Therefore, as confirmed in Experiment 1, Echochocho extract not only has osteoblast proliferation effect, but also promotes calcification of hard tissues, thereby confirming that it can play a large role in fulfilling its function as hard tissues.

Experimental Example 3: Effect of Promoting Proliferation of Human Periodontal Ligament Cells

In order to determine the effect of Eochochocho extract on human periodontal ligament cell proliferation rate, the following experiment was performed.

First, the human periodontal ligament cells were cultured by the following method. The first premolar of a patient admitted for orthodontic treatment was extracted and four times with a biopsy medium consisting of DMEM with 200 unit / ml penicillin, 200 μg / ml streptomycin, and 1 μg / ml amphotericin B. Washed. The periodontal ligament tissue was surgically cut at a point 1/3 from the root to the center and distributed evenly in a 35 mm diameter culture dish, followed by 100 unit / ml penicillin, 100 μg / ml streptomycin and 0.5 μg / ml. The culture medium consisting of DMEM and 10% FBS containing amphotericin B was added and cultured in a 5% CO ₂ air mixed incubator at 37 ° C. and 100% humidity. When the periodontal ligament cells proliferated from the tissue sections and cultured at 3 days intervals, the culture medium was exchanged at 3 days intervals to form a monolayer that completely covered the dish, and then treated with 0.05% trypsin / 0.02% EDTA to separate cells from the culture dish 1: Subcultured at a ratio of 3 to 5-7 generation cells were used in this experiment.

0.5 ml of the culture solution (human containing 1 × 10 ⁴ periodontal ligament cells) was placed in a 24-well culture dish and cell growth rate was measured in the same manner as in Experimental Example 1. The experimental results are shown in Table 3.

TABLE 3

As a result of analyzing the results of the experiment compared to the control group not administered the drug, the standard deviation was large at the time of long-term administration (cell culture 5 days), and no significant difference was found between the experimental data. At 1) it was confirmed that Echochocho extract obtained in Example 1 significantly promoted the proliferation of periodontal ligament cells. Therefore, the hard tissue regeneration accelerator composition of the present invention induces proliferation of periodontal ligament cells, thereby exhibiting the regeneration effect of the lost periodontal ligament cells.

Experimental Example 4 Experimental Effect of Increased Alkaline Phosphatase Activity Using Human Periodontal Ligament Cells

To determine the effect of Echoseongcho extract on the activity of alkaline phosphatase in human periodontal ligament cells, the following experiment was conducted.

Cells were collected and cultured from human periodontal ligament cells in the same manner as in Example 3. The activity of alkaline phosphatase was measured in the same manner as in Example 2 by trypsin treatment to the periodontal ligament cells of humans in the initial dense state. The results were shown in Table 4.

TABLE 4

As a result of analyzing the results of the experiment compared to the control group that did not receive the drug, the standard deviation was large at the time of short-term administration (cell culture 3 days), and therefore, no significant difference was found between the experimental data. In one), it was confirmed that Echochocho extract obtained in Example 1 did not affect the periodontal ligament cell alkaline phosphatase activity.

The therapeutic agent of the present invention promotes the proliferation of osteoblasts and promotes the calcification of hard tissues, thereby promoting hard tissue proliferation and regeneration, as well as the formation of hard tissues having a form of sufficient hardness that can function as hard tissues.

Therefore, the therapeutic agent of the present invention is a natural-derived drug containing an extract obtained from Eoseongcho, which is one of the herbal medicines, and because it has excellent side effect of regenerating and proliferating hard tissue without showing side effects, diseases related to hard tissue regeneration and proliferation, such as osteoporosis and periodontal disease, etc. It provides a hard tissue regeneration accelerator composition that can be used safely in place of existing therapeutic agents for the purpose of prevention and treatment of.

In addition, the therapeutic agent of the present invention can be used as a feed additive of the ovulation livestock used for the purpose of strengthening the egg shell because of its excellent osteoblast proliferation.

Claims (6)

Hard tissue regeneration accelerator composition comprising Eungchocho extract as an active ingredient. The extract of claim 1, wherein the extract is a concentrated solution obtained by filtering the extract obtained by heating the extract and heating it at 40 to 60 ° C. for 5 to 24 hours at 50 to 100 ° C., and then evaporating and condensing the supernatant. Hard tissue regeneration accelerator composition. According to claim 1, Eochocho extract is obtained by cooling the extract obtained by putting the eochochocho into the extraction solvent and warmed at 50 ~ 100 ℃ for 5 to 24 hours by cooling to 40 ~ 60 ℃, and then concentrated by evaporation of the supernatant and dried Hard tissue regeneration accelerator composition, characterized in that the powder. 2. The hard tissue regeneration accelerator composition according to claim 1, wherein the extract is filtered after cooling the edible vinegar in an extraction solvent and cooling the mixture for 5-7 days at room temperature or 4 ° C. The hard tissue regeneration accelerator composition according to claim 1, wherein the extract of Eochochoea is a powder obtained by immersing Echochocho in an extraction solvent, cooling the solution for 5-7 days at room temperature or 4 ° C, and then evaporating and condensing the supernatant. The hard tissue regeneration accelerator composition according to any one of claims 2 to 5, wherein the extraction solvent is an aqueous 50-100% methanol solution.