KR102320679B1 - Composition for preventing or treating osteoporosis comprising complex extract of lycii radicis cortex and achyranthes japonica nakai, and analysis method for standardization thereof - Google Patents

Abstract

The present invention relates to a composition for preventing or treating osteoporosis comprising a complex extract of phalanx phalanx and hyssopus as an active ingredient, and an analysis method thereof, and more particularly, a phalanx phalanx extract and hyssopus extract in a weight ratio of 7:3 to 9:1. It provides a pharmaceutical composition for preventing, improving or treating osteoporosis, a health functional food composition, and a raw material standardization analysis method thereof, containing the alcohol complex extract as an active ingredient.
Accordingly, it is possible to effectively prevent, improve or treat osteoporosis by using the phalangeal skin and hyssopus complex extract mixed in an optimal ratio as a pharmaceutical composition and a health functional food composition, and the active ingredients and content of the phalanx phalanx extract and hyssopus extract by the above analysis method can be standardized.

Description

A composition for preventing or treating osteoporosis comprising a complex extract of phalanx and mussel as an active ingredient, and an analysis method for standardization thereof

The present invention relates to a composition for preventing or treating osteoporosis comprising a complex extract of phalanx phalanx and mussel as an active ingredient, and an analysis method for standardizing raw materials thereof.

Bone tissue is a dense connective tissue surrounded by bone cells and hard calcium tissue around the bone cells, and has a mechanical function of support and muscle attachment. In addition, it is responsible for the function of protecting biological organs and bone marrow and the function of preserving calcium and phosphorus ions to maintain homeostasis. Such bone tissue is composed of a cell matrix such as collagen and glycoprotein and various types of cells such as osteoblasts, osteoclasts, and osteocytes. Osteoblasts derived from bone marrow stromal cells play a major role in bone formation, and osteoclasts derived from hematopoietic stem cells are destroyed and are responsible for resorption of aged bone. Through the balanced action of bone formation and bone resorption, bone remodeling is maintained.

Bone metabolic disease occurs when the balance between osteoblasts and osteoclasts, which play a major role in maintaining bone remodeling, is abnormal. As a representative example of bone metabolic disease, there is osteoporosis. In osteoporosis, the bone resorption activity of osteoclasts increases compared to bone formation by osteoblasts, and as a result, total bone mass decreases, resulting in a fracture even with a slight impact. disease caused by this To date, there is no effective treatment for osteoporosis, and prevention is emphasized.

Osteoblasts responsible for bone formation originate and form from mesenchymal stem cells. Mineralization including calcium formed by osteoblast differentiation can maintain bone strength as well as calcium throughout the body. And it plays a very important function in the homeostasis of hormonal metabolism. Calcium formation by the differentiation of osteoblasts is regulated by vitamin D and parathyroid hormone, and the like, and bone morphogenetic protein (BMP), Wnt, MAP kinase, calcineurin-calmodulin in cells. Alkaline phosphatase (ALP), which is involved in the differentiation of osteoblasts by cross-talk of various signal transduction systems such as kinase (calcineurin-calmodulin kinase), NF-κB, and AP-1, is released in the initial stage of differentiation. After synthesis, osteopontin, osteocalcin (BGLAP gene), type 1 collagen, etc., which are involved in mineralization, are synthesized to form bone by differentiation of osteoblasts. .

Recently, efforts to prevent or treat bone damage by producing osteoblasts in ex vivo culture conditions and using them to strengthen the function of bone tissue are rapidly increasing, and thus various natural products are being studied. However, even if the physiologically active substances of natural products are of the same origin, there may be differences in active ingredients and contents depending on various factors such as growth environment, processing methods, and storage conditions, and thus there may be differences in efficacy. Accordingly, there is a need for a method of standardizing or standardizing so as to consistently exhibit efficacy with uniform active ingredients and content.

Korea Patent Publication No. 10-2016-0098982 (published on August 19, 2016)

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating osteoporosis containing the phalanx phalanx extract and hyssop extract.

The present invention provides a health functional food composition for preventing or improving osteoporosis containing the phalanx phalanx extract and hyssop extract.

In addition, the present invention provides a raw material standardization analysis method of the phalanx phalanx and mussel complex extract.

The pharmaceutical composition for preventing or treating osteoporosis according to the present invention may contain, as an active ingredient, an alcohol complex extract mixed with a phalangeal skin extract and a hyssopus extract in a weight ratio of 7:3 to 9:1.

The health functional food composition for preventing or improving osteoporosis according to the present invention may contain, as an active ingredient, an alcohol complex extract obtained by mixing phalangeal skin extract and hyssopus extract in a weight ratio of 7:3 to 9:1.

The raw material standardization analysis method of the phalanx phalanx and mussel complex extract according to the present invention is to prepare a calibration curve by dissolving and diluting kukoamine B and 20-hydroxyecdysone as standard materials, respectively, in a solvent. step; Preparing an alcohol complex extract by dissolving the phalangeal skin extract powder and the hyssopus extract powder in a solvent and mixing the phalangeal skin extract and the hyssop extract in a weight ratio of 7:3 to 9:1; Compiling a calibration curve by performing HPLC and LC-MS/MS to analyze the content of cucoamine B and 20-hydroxyecdysone in the alcohol complex extract; and comparing the calibration curve of the standard material with the calibration curve for the content of Cucoamine B and 20-hydroxyecdysone in the alcohol complex extract.

The complex extract according to the present invention is mixed in an optimal ratio of phalangeal skin extract and hyssop extract, which can exhibit excellent efficacy in treating osteoporosis, and by using it as a pharmaceutical composition or health functional food composition, it can effectively prevent, improve or treat osteoporosis. .

The analysis method according to the present invention can standardize the active ingredients and contents of the phalangeal skin extract and the hyssop extract, and can be used for quality control. Accordingly, the pharmaceutical composition or health functional food composition containing the complex extract as an active ingredient may exhibit uniform efficacy.

1a is a graph showing alkaline phosphatase (ALP) activity when treated with phalangeal skin extract or mussel extract alone according to an experimental example of the present invention.
Figure 1b is a graph showing alkaline phosphatase (ALP) activity in the case of mixing the phalanx phalanx extract and mussel extract according to an experimental example of the present invention.
Figure 1c is a graph analyzing the cytotoxicity of the extract according to an experimental example of the present invention.
Figure 2a is a graph showing the expression level of Bglap gene mRNA when the phalangeal skin extract or mussel extract according to an experimental example of the present invention is treated alone.
Figure 2b is a graph showing the expression level of Bglap gene mRNA in the case of mixing the phalanx phalanx extract and mussel extract according to an experimental example of the present invention.
Figure 2c is a graph showing the expression level of bone metabolism-related gene markers in the case of mixing the phalangeal skin extract and the mussel extract according to an experimental example of the present invention.
3 is a result of alkaline phosphatase (ALP) staining experiment and mineralization analysis experiment according to an experimental example of the present invention.
Figure 4 is a structure of kukoamine B (kukoamine B), 20-hydroxyecdysone (20-hydroxyecdysone), which are indicators and active ingredients of the phalangeal skin-muscle complex extract according to an embodiment of the present invention.
Figure 5 is a calibration curve compared with the standard of kukoamine B (kukoamine B) and 20-hydroxyecdysone (20-hydroxyecdysone), which are indicators and active ingredients of the phalangeal skin and mussel complex extract according to an embodiment of the present invention. .
6 is a chromatogram of indicators and active ingredients of the phalanx phalanx and mussel complex extract according to an experimental example of the present invention.

Hereinafter, the present invention will be described in detail.

The present inventors confirmed the optimal mixing ratio having efficacy in preventing, improving or treating osteoporosis in the phalanx hyoid complex extract, and analyzed this by HPLC-LC-MSMS method, which has the highest content of kukoamine B (Kukoamine B) And by establishing a raw material standardization method using 20-hydroxyecdysone (20-hydroxyecdysone) of mussel as an index and active ingredient, the present invention was completed.

In the present specification, "phalangeal blood (Latin name: Lycii Radicis Cortex, scientific name: Lycium chinense Miller)" refers to dried root bark of the solanaceae plant Lycium chinense Mill, has a bitter taste and cold properties, and 'goji It can be used interchangeably as 'geunpi', 'bulb root', 'famine', etc. Phalangeal blood is good for cold sweat, seawater, asthma, hematemesis, nosebleeds, urine bleeding, hyperglycemia, and high blood pressure caused by weakness of the body, and is known to be used for neuralgia, headache, shoulder pain, muscle pain, back pain, etc. The pharmacological effects of hypotension and hypoglycemia have been reported.

As used herein, the term "hyssul (Latin name: Achyranthis Radix, scientific name: Achyranthes japonica Nakai)" is the root of the oxus knee, a perennial herbaceous plant belonging to the family Mysaceae, and is called hyssop because its shape is similar to that of a cow's knee. It is known to be effective in treating It is also used a lot when the back and legs feel heavy and painful, and sometimes there are muscle spasms.

As used herein, the term "extract" refers to a substance obtained by extracting a component of a natural product regardless of an extraction method, an extraction solvent, an extracted component, or the form of an extract, and another method after extracting the material obtained by extracting the component of the natural product It may include any material obtainable by processing or processing with

As used herein, the term "complex extract" may include all substances extracted by mixing two or more natural substances or substances obtained by mixing extracts of two or more natural substances, and may be used interchangeably with 'mixed extract'.

As used herein, "prevention" refers to any action that suppresses or delays the onset of osteoporosis disease or at least one or more symptoms of the disease by administration of the pharmaceutical composition or health functional food composition according to the present invention. Also included is treatment of a subject in remission of the disease to prevent or prevent recurrence.

As used herein, "treatment" refers to any action that improves or advantageously improves osteoporosis disease or at least one or more symptoms of the disease by administering the pharmaceutical composition according to the present invention, such as alleviating, reducing, or disappearing the symptoms. do.

As used herein, "improvement" refers to any act of improving or beneficially changing osteoporosis disease or at least one symptom of the disease by ingestion of the health functional food composition according to the present invention, such as alleviation, reduction, or disappearance of the symptom. means

As used herein, the term "pharmaceutical composition" refers to a composition administered for a specific purpose, and for the purpose of the present invention, it means to be administered to prevent or treat osteoporosis disease or at least one or more symptoms of the disease.

As used herein, "health functional food" is a food manufactured and processed using raw materials or ingredients useful for the human body, and is a food with high medical and medical effects processed to efficiently exhibit bioregulatory functions in addition to nutrition supply. meaning and may be used interchangeably with terms known in the art, such as functional food.

The present invention provides a pharmaceutical composition for preventing or treating osteoporosis containing the phalanx phalanx and mussel complex extract as an active ingredient.

In the pharmaceutical composition according to the present invention, the phalanx phalanx and hyssul complex extract may include an alcohol complex extract in which the phalangeal skin extract and hyssopus extract are mixed in a weight ratio of 7:3 to 9:1, preferably 8:2, The alcohol complex extract may be extracted with an aqueous ethanol solution of 60 to 80% by weight, preferably 70% by weight.

The composition can promote the differentiation of osteoblasts and can promote the mineralization of osteoblasts, and thus can be used as an active ingredient in a pharmaceutical composition for preventing or treating osteoporosis. According to an experimental example of the present invention, when the phalangeal skin extract and the hyssopus extract were mixed in a weight ratio of 8:2 and treated with osteoblasts, alkaline phosphatase (ALP) activation related to the differentiation of osteoblasts was the highest. , the expression levels of all three bone metabolism-related gene markers (Alp, Runx2, Bglap) were significantly increased, and the number of cells stained by ALP and the number of cells stained by nodule formation (mineralization) were further increased. More details will be described later by the following experimental examples.

The pharmaceutical composition according to the present invention may be prepared according to a conventional method in the pharmaceutical field.

The pharmaceutical composition according to the present invention may be combined with an appropriate pharmaceutically acceptable carrier according to the formulation, and if necessary, excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. can be manufactured. The "pharmaceutically acceptable" means that it is not toxic to cells or humans exposed to the pharmaceutical composition, and the appropriate carrier does not inhibit the activity and properties of the extract according to the present invention, the dosage form and It may be selected differently depending on the formulation.

The pharmaceutical composition according to the present invention may be applied in any dosage form, and more specifically, it may be formulated and used in parenteral dosage forms of oral dosage forms, external preparations, suppositories and sterile injection solutions according to conventional methods.

The solid dosage form among the oral dosage forms is in the form of tablets, pills, powders, granules, capsules, etc., and at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may be included. In addition, the capsule formulation may further include a liquid carrier such as fatty oil in addition to the above-mentioned substances.

Among the oral dosage forms, liquid formulations include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. have.

The parenteral formulation may include a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried formulation, and a suppository. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, and the like can be used. Without being limited thereto, any suitable agent known in the art may be used.

In addition, the pharmaceutical composition according to the present invention _{may further add calcium or vitamin D 3} and the like to enhance therapeutic efficacy.

In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be administered in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects.

The effective dose level of the pharmaceutical composition is determined by the purpose of use, the age, sex, weight and health status of the patient, the type of disease, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment It may be determined differently depending on factors including the duration, formulation or concurrent use of drugs and other factors well known in the medical field. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, may be administered once to several times a day. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition according to the present invention may be administered to any animal that can develop osteoporosis, and the animal may include, for example, not only humans and primates, but also livestock such as cattle, pigs, horses, and dogs.

The pharmaceutical composition according to the present invention may be administered by an appropriate administration route according to the form of the formulation, and may be administered through various routes, either oral or parenteral, as long as it can reach the target tissue. The administration method is not particularly limited, and for example, oral, rectal or intravenous, intramuscular, subcutaneous, endobronchial inhalation, intrauterine dural or intracerebroventricular injection, etc. can be administered in a conventional manner. .

The pharmaceutical composition according to the present invention may be used alone for preventing or treating osteoporosis, or may be used in combination with surgery or other drug treatment.

The present invention provides a health functional food composition for preventing or improving osteoporosis containing the phalanx phalanx and mussel complex extract as an active ingredient.

In the health functional food composition according to the present invention, the phalanx phalanx and hyssul complex extract may include an alcohol complex extract mixed with phalangeal skin extract and hyssopus extract in a weight ratio of 7:3 to 9:1, preferably 8:2. And, the alcohol complex extract may be extracted with an aqueous ethanol solution of 60 to 80% by weight, preferably 70% by weight.

The composition can promote the differentiation of osteoblasts and can promote the mineralization of bone cells, and can be used as an active ingredient of a health functional food composition for preventing or improving osteoporosis. According to an experimental example of the present invention, when the phalangeal skin extract and the hyssopus extract were mixed in a weight ratio of 8:2 and treated with osteoblasts, alkaline phosphatase (ALP) activation related to the differentiation of osteoblasts was the highest. , the expression levels of all three bone metabolism-related gene markers (Alp, Runx2, Bglap) were significantly increased, and the number of cells stained by ALP and the number of cells stained by nodule formation (mineralization) were further increased. More details will be described later by the following experimental examples.

In the health functional food composition according to the present invention, the health functional food may be prepared in powder, granule, tablet, capsule, syrup or beverage, etc., and there is no limitation in the form that the health functional food can take, It can include any food with meaning. For example, beverages and various drinks, fruits and their processed foods (canned fruit, jam, etc.), fish, meat and their processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ), etc. are possible, and may include all functional foods in a conventional sense. It may also include food used as feed for animals.

The health functional food composition according to the present invention may be prepared by further including pharmaceutically acceptable food additives and other suitable auxiliary ingredients commonly used in the art. For example, flavoring agents, natural carbohydrates, sweetening agents, vitamins, electrolytes, coloring agents, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents, etc. can In particular, as the natural carbohydrate, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol can be used. , as the sweetener, natural sweeteners such as taumatine and stevia extract or synthetic sweeteners such as saccharin and aspartame may be used.

The health functional food composition according to the present invention has the advantage that there are no side effects that may occur during long-term administration of the drug using food as a raw material, unlike general drugs, and has excellent portability, so that it can be ingested as an adjuvant for preventing or improving osteoporosis. can

In addition, the present invention provides a raw material standardization analysis method of the phalanx phalanx and mussel complex extract.

The raw material standardization analysis method according to the present invention comprises the steps of dissolving kukoamine B and 20-hydroxyecdysone as standard materials in a solvent, respectively, and diluting them to prepare a calibration curve; Preparing an alcohol complex extract by dissolving the phalangeal skin extract powder and the hyssopus extract powder in a solvent and mixing the phalangeal skin extract and the hyssop extract in a weight ratio of 7:3 to 9:1; Compiling a calibration curve by performing HPLC and LC-MS/MS to analyze the content of cucoamine B and 20-hydroxyecdysone in the alcohol complex extract; and comparing the calibration curve of the standard material with the calibration curve for the content of Cucoamine B and 20-hydroxyecdysone in the alcohol complex extract.

In the calibration curve preparation step, the standard material, kukoamine B, is an indicator and active ingredient of the phalanx, and the 20-hydroxyecdysone, which is a kind of ecdysteroid, is mussel. Indices and active ingredients of, but the standard material is not limited thereto.

In the preparation step of the phalanx phalanx and hyssul complex extract, the phalanx phalanx and hyssul complex extract may be prepared by mixing the phalangeal skin extract and hyssul extract at a weight ratio of 7:3 to 9:1, preferably 8:2.

In the step of preparing the calibration curve of the phalanx phalanx and mussel complex extract, the content of cucoamine B and 20-hydroxyecdysone in the alcohol complex extract can be derived by analyzing the chromatogram obtained by performing HPLC and LC-MS/MS. can

The analysis method according to the present invention may further include a validation step including at least one item selected from the group consisting of specificity, linearity, range, accuracy, precision, and system suitability, and through the verification step, the analysis Methods can be evaluated and reviewed.

In the verification step, the specificity is for verifying that only the index component can be clearly identified. According to an experimental example of the present invention, the index component is the index component through the separated peak of the chromatogram. Coamine B and 20-hydroxyecdysone can be identified.

The linearity and range are the ability to obtain an analysis result that is directly proportional to the concentration (amount) of an indicator component in a sample in a given range, and can be visually evaluated through a calibration curve according to an experimental example of the present invention. The validity of the range can be verified by obtaining a linear correlation coefficient (R ^{2 ) from the calibration curve.}

The accuracy is evaluated from the results of repeated measurements at least three times for each concentration for at least three concentrations including a prescribed range, and can be reviewed as a recovery rate (%).

The precision (precision) is evaluated from the results of repeated measurements at least 3 times for each concentration for 3 concentrations including the prescribed range, and can be reviewed with the relative standard deviation (coefficient of variation).

The system suitability may be evaluated by parameters such as a tailing factor, a capacity factor, theoretical plates, and resolution.

According to the analysis method according to the present invention, the content of the cucoamine B is 5.2 to 7.8 parts by weight, and the 20-hydroxyecdysone of the phalangeal skin-muscle complex extract is based on 100 parts by weight of the total alcohol complex extract. The content may be 0.184 to 0.276 parts by weight. It is possible to standardize the active ingredient of the phalanx phalanx and mussel complex extract within the above range.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Example 1> Preparation of phalangeal skin extract and hyssop extract

After shaking the phalanges and mussels in 70% ethanol aqueous solution for 5 hours, they were left for about 20 hours, and then filtered with filter paper. Each extract powder was prepared by freeze-drying the filtrate. The mixed extract of phalanx phalanx and wooseul was prepared by mixing phalanx extract powder and hyssop extract powder in a weight ratio of 8:2.

<Experimental Example 1> Cell culture

C3H10T1/2 cells, which are mouse mesenchymal stem cells, and MC3T3-E1 cells, which are osteoblasts, were treated with 10% Fetal Bovine Serum (FBS), 1mM Sodium Pyruvate, 100unit/L Penicillin, and 100 Incubated in basal medium Eagle (BME) and α-minimun essential medium (α-MEM) supplemented with mg/L streptomycin at 37°C and 5% CO ₂ conditions. did. In the case of MC3T3-E1 cells, the osteoblast differentiation inducing agents ascorbic acid (50ug/ml) and β-glycerol phosphate (β-Glycerophosphate, 10mM) were added to induce differentiation for 3 days, and then, phalanx phalanx and mussel extract Single or mixed extracts were added and further incubated for 48 hours. Cells cultured in the untreated state were used as a control.

<Experimental Example 2> Alkaline phosphatase (ALP) activity analysis

After culturing the experimental group and the control group (untreated) for 48 hours with the phalangeal skin extract and the hyssopus extract prepared according to Example 1 alone or mixed, the alkaline phosphatase (Alkaline phosphatase; hereafter referred to as ALP) assay kit (ALP Assay Kit) was used to measure ALP enzyme activity. After washing the cells with physiological saline, the cells lysed using a cell lysing agent were treated with p-nitrophenylphosphate, a substrate of ALP, and incubated at 37° C. for 1 hour. After adding 0.5N NaOH as a reaction stop solution, absorbance was measured at a wavelength of 405 nm.

1a is a graph showing alkaline phosphatase (ALP) activity when the phalangeal skin extract or mussel extract according to an experimental example of the present invention is treated alone.

Referring to Figure 1a, the graph on the left shows the ALP activity when the phalangeal skin extract (LRC) is treated alone, the graph on the right shows the ALP activity when the hyssopus extract (AJ) is treated alone, and the phalangeal skin extract or the hyssopus extract is treated with 2 , when MC3T3-E1 cells, which are osteoblasts, were treated at concentrations of 10 and 50 μg/ml, the best ALP activity was shown in both extracts at 10 μg/ml (*: P < 0.05 VS. Control).

Figure 1b is a graph showing alkaline phosphatase (ALP) activity in the case of mixing the phalanx phalanx extract and mussel extract according to an experimental example of the present invention.

Referring to Figure 1b, the phalangeal skin extract (LRC, 10μg / ml), hyssop extract (AJ, 10μg / ml) and the two extracts are mixed at a ratio of 9:1, 8:2 and 7:3 (LRC: AJ) When the cells were treated with phalanx, the best ALP activity was shown in an 8:2 mixture of phalangeal skin extract and hyssopus extract. (* : P < 0.05 VS. Control)

<Experimental Example 3> Cell proliferation assay

Cell proliferation assay was measured using a cell viability assay kit (EZ-Cytox Enhanced Cell Viability Assay Kit) using Water Soluble Tetrazolium Salts (hereinafter, WSTs).

Specifically, cells (3×10 ³ cells/well) were inoculated in a 96-well plate and _{cultured for 24 hours in a growth medium at 37° C. supplied with 5% CO 2} , and then the phalangeal skin extract and hyssopus extract alone or mixed to the cells. treated and further incubated for 48 hours. WSTs were treated with cultured cells and samples and incubated at 37°C for 2 hours. Absorbance was measured at 450 nm using 655 nm as the reference wavelength.

Figure 1c is a graph analyzing the cytotoxicity of the extract according to an experimental example of the present invention.

Referring to Figure 1c, osteoblastic MC3T3-E1 cells were analyzed for cytotoxicity after treatment with phalangeal skin extract, hyssopus extract alone and a mixture of the extracts. As a result, the treatment of the extract does not affect cell proliferation. appear.

<Experimental Example 4> Analysis of osteoblast differentiation gene marker expression level

Alp (Alkaline phosphatase), Runx2 (Runt-related transcription factor 2), Bglap (bone gamma-carboxyglutamate protein), (Osteocalcin, Ocn) among the bone metabolism-related gene markers showing changes in expression level during osteocytic differentiation The gene expression level was confirmed by real-time PCR.

After harvesting the cells of the experimental group treated by mixing the control cells (untreated) with the phalanx extract and the hyssopus extract, total RNA was isolated using Trizol, and DNA was digested to remove genomic DNA. Enzyme I (DNase I) was reacted at room temperature for 15 minutes and treated with ethylenediaminetetraacetic acid (EDTA) to inactivate DNA degrading enzyme I. Thereafter, cDNA was synthesized by inducing a reverse transcription reaction using an oligo dT primer as a primer, and then real-time PCR was performed using sequence-specific primers of each gene and SYBR Green. The expression level was analyzed using a relative quantification method of the target gene using Gapdh (Glyceraldehyde 3-phosphate dehydrogenase) gene as a control. The gene-specific primer sequence information used in this method is shown in Table 1 below.

Table 1 below is primer sequence information for confirming the expression level of the gene marker used to determine the degree of osteocytic differentiation.

Kinds gene sequence name Sequence information (5'→3') control
genetic markers
Gapdh Gapdh-F TGA CCA CAG TCC ATG CCA TC Gapdh-R GAC GGA CAC ATT GGG GGT AG

osteoblasts
Differentiation measurement
genetic markers Bglap
(Osteocalcin) Bglap-F TAG TGA ACA GAC TCC GGC GCT A Bglap-R TGT AGG CGG TCT TCA AGC CAT
Alp Alp-F TCC CAC GTT TTC ACA TTC GG Alp-R CCC GTT ACC ATA TAG GAT GGC C
Runx2 Runx2-F TAA AGT GAC AGT GGA CGG TCC C Runx2-R TGC GCC CTA AAT CAC TGA GG

Figure 2a is a graph showing the expression level of Bglap gene mRNA when the phalangeal skin extract or mussel extract according to an experimental example of the present invention is treated alone.

Referring to Figure 2a, when MC3T3-E1 osteoblasts were treated with 10, 20 μg/ml of phalangeal skin extract (LRC), 10, 20 μg/ml of mussel extract (AJ), the expression level of Bglap gene mRNA compared to the untreated control group was significantly increased, and the expression level was high when the concentration of the phalangeal skin extract was 10μg/ml and the concentration of the hyssopus extract was 20μg/ml. (*: P < 0.05 VS. Control)

Figure 2b is a graph showing the expression level of Bglap gene mRNA in the case of mixing the phalanx phalanx extract and mussel extract according to an experimental example of the present invention.

Referring to FIG. 2b, when the phalangeal skin extract and the hyssopus extract 10 and 20 μg/ml concentrations were mixed in a ratio of 9: 1, 8: 2 and 7: 3, respectively, and treated with MC3T3-E1 osteoblasts, the concentration of 20 μg/ml When each extract was mixed in a ratio of 8:2, the best expression of the Bglap gene was exhibited, and the Bglap gene expression level was increased by about 50% compared to the single treatment of the phalanx phalanx extract or the hyssop extract. (* : P < 0.05 VS. Control)

Figure 2c is a graph showing the expression level of bone metabolism-related gene markers in the case of mixing the phalangeal skin extract and the mussel extract according to an experimental example of the present invention.

Referring to FIG. 2c, C3H10T1/2 mesenchymal stem cells and MC3T3-E1 osteoblasts were treated with an extract obtained by mixing phalanx phalanx extract and hyssopus extract at a concentration of 20 μg/ml in an 8:2 ratio to treat three types of bone metabolism-related gene markers (Alp). , Runx2, Bglap), the expression levels of all three gene markers were significantly increased in C3H10T1/2 cells and MC3T3-E1 cells, and more specifically, about 80~ It was found that the expression level was increased by 170% (* : P < 0.05 VS. Control).

<Experimental Example 5> ALP staining and mineralization analysis of osteoblasts

For the ALP staining experiment, mouse osteoblasts, MC3T3-E1 cells, were treated with a phalangeal skin extract at a concentration of 20 μg/ml each in a 48-well plate, a hyssopus extract, and an extract obtained by mixing the extract in an 8:2 ratio, 37 ℃, 5% for 48 hours. Incubated under CO _{2 conditions.} After washing the cells with a phosphate buffer (PBS buffer), the cells were stained with an ALP reagent kit, and the number of ALP-stained cells was observed under a microscope.

In the mineralization assay, mouse osteoblasts, MC3T3-E1 cells, were treated with a phalangeal skin extract, a mussel extract, and an extract (20 μg/ml concentration each) mixed in a 48-well plate at an 8:2 ratio (each concentration of 20 μg/ml) at 37° C., 5 % CO ₂ Incubated under conditions. The cell culture medium was changed every 3 days. After washing the cells with a phosphate buffer solution, the cells were fixed with 70% ethanol, treated with 40 mM Alizarin red S, and the number and extent of stained cells were observed under a microscope.

3 is a result of an ALP staining experiment and mineralization analysis experiment according to an experimental example of the present invention.

Referring to Figure 3, in the case of the ALP staining experiment, compared to the single treatment of the phalangeal skin extract or the hyssopus extract, it was found that the number of ALP-stained cells was further increased when the extract mixed with the phalangeal skin extract and the hyssop extract was treated at 8:2.

In addition, in the case of the mineralization analysis experiment, compared to the single treatment of the phalangeal skin extract or the hyssopus extract, Alizarin less S staining by nodule formation (mineralization) during the treatment of the extract mixed with the phalangeal skin extract and the hyssop extract at 8:2 It was found that the number of cells increased further.

<Experimental Example 6> Analysis of index and active ingredient content of phalanx phalanx and mussel complex extract using HPLC-UV analysis

1. Material

Kukoamine B (kukoamine B) standard, 20-hydroxyecdysone (20-hydroxyecdysone) standard, phalangeal skin extract (powder) and hyssop extract (powder) were prepared.

Figure 4 is a structure of kukoamine B (kukoamine B), 20-hydroxyecdysone (20-hydroxyecdysone), which are indicators and active ingredients of the phalangeal skin-muscle complex extract according to an embodiment of the present invention.

2. Pretreatment

25% methanol containing 0.2% formic acid [hereinafter, 25% MeOH (0.2% Formic acid)], and the same amount of each standard was taken to be 40 μg/mL and 2 μg/mL. This was diluted with 25% MeOH (0.2% Formic acid) and kukoamine B was 20, 16, 10, 5, 2, 1 μg/mL, and 20-hydroxyecdysone was 1 , 0.8, 0.5, 0.25, 0.1, and 0.05 μg/mL.

After finely grinding and homogenizing the phalangeal skin extract powder and the hyssopus extract powder, each powder is weighed and dissolved in 25% MeOH (0.2% Formic acid) solvent to obtain a final concentration of 160μg/mL for the phalange extract and 40μg/mL for the hyssop extract. was made to become After mixing the phalanx phalanx extract powder and mussel extract powder, 2 g was weighed and sufficiently dissolved in 5 mL of 25% MeOH (0.2% Formic acid), and filtered. This was diluted with a solvent of 25% MeOH (0.2% Formic acid) to a final concentration of 200 μg/mL.

3. Analysis conditions

3-1. Ultra Performance Liquid Chromatography (UPLC, Acquity UPLC, Waters)

Column: Kinetex® EVOC18 (150×2.1mm, 2.6μm)

Column oven: 35°C

Injection volume: 1 μL

Flow rate: 0.6mL/min

Wavelength: 254nm

Mobile phase A: 0.2% Formic Acid in DW, B: 0.2% Formic Acid in Acetonitrile

Table 2 below shows the gradients used in this analysis.

A B 0 95 5 3 95 5 19 80 20 19.1 5 95 23 5 95 23.1 95 5 25 95 5

3-2. Quadrupole mass, SQ detector, Waters

Ion source: ES-

Data type: SIR

Capillary voltage: 3.5V

Source temperature: 150°C

Desolvation temperature: 350°C

Desolvation Gas Flow: 550L/hr

4. Analysis method

First, 1 μL of each sample is injected into HPLC and LC-MSMS (Liquid Chromatography tandem-mass spectrometry, SIR mode) equipment to compare the standard material and a calibration curve, and the phalanx pelvis and musculature complex extract is injected into HPLC and LC-MSMS for analysis It was quantified by checking the peak height of the chromatogram for one indicator and active ingredient.

Figure 5 is a calibration curve compared with the standard of kukoamine B (kukoamine B), 20-hydroxyecdysone (20-hydroxyecdysone), which are indicators and active ingredients of the phalangeal skin and mussel complex extract according to an experimental example of the present invention. , Figure 6 is a chromatogram of the index and active ingredient of the phalanx phalanx and mussel complex extract according to an experimental example of the present invention.

Table 3 is a validation item for the analysis method of the phalanx phalanx and mussel complex extract according to an experimental example of the present invention, and the specificity, linearity, range, and accuracy of the content analysis method ( The evaluation criteria of accuracy, precision, and system suitability were established.

validation
item Criteria kukoamine B
Validation Results 20-hydroxyecdysone
Validation Results

specificity 1) Non-detection of index component peaks in diluents and mobile phases
2) Component peak detection in the sample solution and the standard solution at the same time
3) When comparing the molecular weights of the sample solution and the standard solution, they are consistent 1) Peak non-detection

2) Peak detection at the same time

3) Match molecular weight 1) Peak non-detection

2) Peak detection at the same time

3) Match molecular weight linearity
&
range 1) Scope
2) R ² ≥0.99
3) no visually convex parts 3.75 ~ 90㎍/㎖
0.9993 to 0.9998
None/None 0.0625 ~ 1.5㎍/㎖
0.9998 to 0.9999
None/None

accuracy 1) Recovery rate of content at low concentration
: 100 ± 7%
2) Recovery rate of content at medium concentration
: 100 ± 7%
3) Recovery rate of content at high concentration
: 100 ± 7%
99.50 ~ 105.1%

100.3 to 100.5%

102.5 to 106.3%
100.7 to 102.0%

101.3 ~ 103.8%

102.0 ~ 103.4%



precision 1) Repeatability
% RSD ≤ 3.0% of area value at low concentration
% RSD ≤ 3.0% of area value at intermediate concentration
% RSD ≤ 3.0% of area value at high concentration
2.82
1.79
1.64
1.66
0.69
0.54 2) Daily repeatability
% of content at medium concentration Maximum RSD ≤ 3.0%
2.59
1.85 3) Indoor precision
% RSD ≤ 5.0% of Investigator A concentration
% RSD ≤ 5.0% of Investigator B Concentration
2.59
2.47
0.63
0.86 system
compatibility
1) RT % RSD ≤ 2.0%
1.31
0.73

5. Determination of index and active ingredient content of phalangeal skin and mussel complex extract using HPLC-UV analysis

Table 4 shows the index and content of active ingredients of the raw material standardized phalangeal skin-muscle complex extract according to an experimental example of the present invention.

Sample name kukoamine B 20-hydroxyecdysone content RSD (%) content RSD (%) Phalangeal Skin and Urseul Mixed Powder 5.2~7.8% 0.807 0.184 to 0.276% 2.248

Referring to Table 4, the content of kukoamine B of the phalangeal skin-muscle complex (8:2) extract determined using HPLC-UV analysis is in the range of 5.2 to 7.8%, and 20-hydroxyex The content of disone (20-hydroxyecdysone) was confirmed within the range of 0.184 to 0.276%.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

delete delete delete delete delete preparing a calibration curve by dissolving kukoamine B and 20-hydroxyecdysone as standards, respectively, in a solvent and diluting them;
Dissolving the phalanx phalanx extract powder and hyssop extract powder in a solvent to prepare an alcohol complex extract mixed with phalangeal skin extract and hyssop extract in a weight ratio of 7:3 to 9:1;
Compiling a calibration curve by performing HPLC and LC-MS/MS to analyze the content of cucoamine B and 20-hydroxyecdysone in the alcohol complex extract; and
Comparing the calibration curve of the standard material and the calibration curve for the content of cucoamine B and 20-hydroxyecdysone in the alcohol complex extract; raw material standardization analysis method of the phalanx pelvis and mussel complex extract comprising a. 7. The method of claim 6,
Based on 100 parts by weight of the total alcohol complex extract,
The content of the cucoamine B is 5.2 to 7.8 parts by weight,
The content of the 20-hydroxyecdysone is 0.184 to 0.276 parts by weight, characterized in that the raw material standardization analysis method of the phalanx hyssopus complex extract. 7. The method of claim 6,
The analysis method is
Specificity, linearity, range, accuracy, precision, and method for standardization analysis of raw materials of a complex extract of phalanx, characterized in that it further comprises a validation step including one or more items selected from the group consisting of precision and system suitability.

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