KR100911672B1 - Health food composition for strengthening bone matrix and method for separating components for strengthening bone matrix - Google Patents

Abstract

A health food composition for strengthening bone matrix and a method for separating a bone matrix strengthening component are provided to inhibit the differentiation of osteoclast and to prevent bone destruction using Carthamus seed extract. A health food composition for strengthening bone matrix is composed of tracheloside or N-(p-coumaroyl) serotonin mono-beta-D-glucopyranoside as an active ingredient and one or more materials selected from the group consisting of dried Rehmannia root extract, Ganoderma lucidum extract, Acanthopanax senticosus extract, soybean extract, pomegranate extract, burdock extract, cinnamon extract, Ca, P, Mg, casein phosphopeptide and vitamin D3. The health food composition for strengthening bone matrix has a liquid or solid phase. When the health food composition for strengthening bone matrix is a liquid, sucrose fatty acid ester is used for an improvement in dispersibility.

Description

Health food composition for strengthening bone matrix and method for separating components for strengthening bone matrix}

The present invention relates to a bone food fortifying health food composition and a method for separating bone mineral fortifying components, and more particularly, a bone food fortifying health food composition containing a horam extract, a liquid preparation for bone mineral fortifying health food containing the composition or a solid. The present invention relates to a method for separating bone matrix enhancing active substances from preparations and homogenized extracts.

Horamhwa is an annual herb belonging to the Asteraceae family, and is a medicinal plant that has been cultivated for a long time in Korea, China, and Japan, and is known to be effective in tong economy, fish blood and bone mineralization. As for bone reinforcement, foreign interests such as China are being distributed to the market at the same time. However, the prevention and treatment of osteoporosis has been known as folk word of mouth, and there is no scientific and specific information.

In addition, since the components involved in bone matrix strengthening activity is not known, only simple extraction products are on the market rather than technologies and products using active ingredients. Considering that current chemotherapy supplements only partial relief but shows limitations in fundamental treatments such as accompanying side effects, homing is considered to be of significance as a preventive material.

Therefore, the present invention relates to the development of bone matrix strengthening products without side effects by participating in the domestic and overseas market for the prevention and treatment of bone matrix strengthening up to 300 billion, 10 billion dollars abroad.

The present invention has been made in accordance with the requirements as described above, the present invention is to develop a health food composition for horamyeo bone substrate strengthening through the multi-faceted experiments using domestic hohwa and to develop a technology for separating, purifying, and identifying bone matrix fortifying active substances It is. The bone matrix strengthening composition and core materials will be used in various ways as food materials for improving bone diseases, health functional foods, useful bone-related substances, and pharmaceutical materials, and contribute to well-being and LOHAS society in an aging society.

In order to solve the above problems, the present invention provides a health food composition for strengthening bone matrix containing a water or methanol extract of the homogenized.

In addition, the present invention provides a liquid formulation or a solid formulation for health food for strengthening bone matrix containing the composition.

In addition, the present invention provides a method for separating the bone matrix strengthening active substance from the horamyeo extract.

According to the present invention, by inventing a method for isolating, purifying, and identifying a bone matrix strengthening active ingredient whose safety and activity has been proven to be safe from horamification, as well as using a bone health single ingredient, bone matrix strengthening activity using a homogenized or homogenized extract It can be used for medicines and foods. In addition, the composition and the component material containing the homing bone matrix strengthening component does not show cytotoxicity at most concentrations as a result of the bone strengthening activity search, and exhibits a high value in Alkaline phosphatase (ALP) activity, which is an index of bone matrix strengthening. In addition, osteoclasts inhibit differentiation, promote bone formation, and suppress bone destruction. Hormonal osteoclast activity extracts remain relatively active at temperature and pH changes, suggesting the possibility of action of the active ingredient in the body and processing.

In order to achieve the object of the present invention, the present invention provides a health food composition for strengthening bone matrix containing a water or methanol extract of the Horam.

The health food composition for strengthening bone matrix of the present invention is added to plant extracts such as raw yellow grass, ganoderma lucidum, prickly pear, soybean, pomegranate, burdock, broiler extract, Ca, P, Mg, CPP (casein phospho peptide), vitamin D _3, etc. It may contain, but is not limited thereto, and may contain various plant extracts that may be used in food compositions in addition to the aforementioned plant extracts. Preferably, the healthy food composition for strengthening bone matrix of the present invention is selected from the group consisting of raw yellow sulfur, ganoderma lucidum, soybean, soybean, pomegranate, burdock, broiler, Ca, P, Mg, casein phospho peptide (CPP) and vitamin D ₃ It may further contain one or more of which is most preferably, further contains raw turmeric, ganoderma lucidum, soybean, pomegranate, burdock, broiler, Ca, P, Mg, casein phospho peptide (CPP) and vitamin D ₃ can do. Prickly Pear Extract is a universal medicine for relieving physical and mental stress. It also has erectile dysfunction, afterlife, elimination of customs, muscle skeletal strengthening effect and superior adaptogen activity than ginseng to enhance nonspecific bio-resistance and control pathology process. It is well known that it has a normalizing effect and is widely used as a raw material of Chinese medicine and food.

In the health food composition for strengthening bone matrix of the present invention, the active ingredient of the horamophilic extract is trakiloside or N- ( p -coumaroyl) serotonin mono- β -D-glucopyranoside, and the trakiloside or N- ( p -coumaroyl) serotonin mono- β -D-glucopyranoside is characterized by activating the osteoblast system and inhibiting the osteoclast system.

According to ALP activity-guided fractionation from methanol extracts of the Horam Seeds, it was identified the tracheoside and the N- (p-coumaroyl) serotonin mono-β-D- glucopyranoside bone matrix strengthening active substance. Hormone-rich bone matrix-enhancing actives containing the above components did not show cytotoxicity in human-like cell lines. Alkaline phosphatase (ALP) activity as an indicator of bone activity was up to 30% or more compared to the control. Horamophilic extracts exhibited both osteogenic and osteoclast inhibitory properties, and their inhibitory activity against differentiation of multinucleate cells was higher than that of RANKL and OPG. To cross-verify bone matrix strengthening activity, we used a cockerel test system for bone matrix formation and an ovarian ablation mouse model for female hormone changes. Hormone samples were not toxic in liver, kidney, and glucose metabolism, increased bone strength, and were shown to aid bone matrix and bone mineral formation.

The composition may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents, etc., tablets, capsules, powders, granules, suspensions, emulsifiers, syrups, solutions or It may be formulated in a unit dosage form or in multiple dosage form pharmaceutical preparations, such as preparations for parenteral administration.

The present invention also provides a liquid formulation for bone matrix strengthening health food containing the health food composition for bone matrix strengthening according to the present invention. Sucrose fatty acid ester may be used to improve the dispersibility of the aqueous solution of the liquid formulation.

Liquid formulations of the present invention may be in the form of a beverage, such as pouches, cheer packs or bottles, but is not limited thereto.

The oxidative stability measured for processing material of Horamification was effective in TBHQ and tocopherol among BHA, BHT, PG, α-tocopherol and TBHQ. Carrageenan, sucrose fatty acid ester and the like to improve the dispersibility. To solve the problem of high precipitation rate in aqueous solution, arabic gum, carrageenan, and sucrose fatty acid esters were added up to 0.1% to 0.7%, and the optical dispersibility was measured using Turbiscan for the first time.

The present invention also provides a solid preparation for bone food strengthening health food containing the health food composition for bone matrix strengthening according to the present invention.

The solid preparation of the present invention may be in the form of capsules, pills, tablets, and the like, but is not limited thereto. In addition to mobility, portability, and dietary convenience, solid workpieces have been developed to enhance their utilization as processed materials. The homing bone matrix strengthening composition showed stability without loss of bone matrix strengthening activity at temperature and pH changes, thereby indirectly confirming stability in processing and body.

The present invention also provides

Vacuum concentrating the homogenized extract obtained by extracting with water or methanol in the homogenized solution;

Dispersing the concentrate of the homogenized extract in a mixed solvent of water and ethyl acetate to form a water layer and an ethyl acetate layer, dispersing butanol in the water layer, and then separating the solvent;

Concentrating the solvent layer in vacuo and purifying by performing column chromatography from a butanol layer and an ethyl acetate layer; And

It provides a method for separating the bone matrix strengthening active material from the Horamyeo extract comprising the step of identifying the compound by analyzing the spectral data of NMR, MS, IR of the extract obtained by the purification.

The active ingredient for strengthening the bone matrix separated and purified by the method of the present invention is characterized in that it is trachiloside or N- ( p -coumaroyl) serotonin mono- β -D-glucopyranoside.

Isolation, Purification, and Identification of Hormone Bone Substrates Enhancement Components Substances were fractionated from the homogenized extracts using polar solvents, and bone matrix enhanced active components were isolated using human-like bone cell systems as a test system. Separation, purification, and identification of the bone matrix strengthening component of Horamification were carried out in the order of extraction concentration, solvent fractionation, column chromatography, purification, and identification. Horamification was extracted with water and methanol 2: 8 solvent and concentrated by vacuum evaporation. It was. The sample concentrate was dispersed in a mixed solvent of water and ethyl acetate to form a water layer and an ethyl acetate layer. Subsequently, butanol was added to the water layer and dispersed, and finally, solvent separation was achieved in three layers, an ethyl acetate layer, a butanol layer, and a water layer. Each solvent layer was concentrated by vacuum evaporation and tested for bone formation ability by concentrate. One lignan compound and one alkaloid compound were isolated and identified according to the ALP activity-guided fractionation from the MeOH extracts of the Horamea seeds, which are the tricheloxide and N- (p-coumaroyl) serotonin mono- β-D-glucopyranoside.

Supercritical extraction can be performed to diversify the extraction method of bone matrix strengthening active ingredient. After the raw material was placed in a cylinder, supercritical carbon dioxide was extracted by rotating the CO _{2 up} to 2,500 L at 50 ° C and 5000 psi, which was excellent in reducing solvent recovery costs and preserving ingredients.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example

Example 1 Isolation, Purification, Identification of Bone Matrix Enhancers

3.8 kg of dry fine hoarse (also known as safflower) seeds contain 80% aq. MeOH was added and extracted three times a day at room temperature while shaking. The extract was concentrated under reduced pressure to obtain 237 g of MeOH extract, which was suspended by adding 2 L of distilled water, and then partitioned with solvent in the same amount of EtOAc and n -BuOH to obtain EtOAc (54 g), n -BuOH and a water fraction (FIG. 1). ). For the analysis of the material profile of the eosinophilic extract, each solvent fraction was concentrated and developed by thin layer chromatography (TLC), followed by qualitative analysis (Dragendorff's reagent, Ferric chloride, 10% H ₂ SO _4, etc.). The profile was qualitatively analyzed.

1.1) Proliferation and Bone Substrate Enhancement Activity of Solvent Fraction

The cell proliferation ability and ALP activity of solvent fractions, EtOAc, BuOH were investigated as a basis for the search for the bone matrix enhancing active ingredient of Horamation. The EtOAc fraction showed the highest proliferative activity at 150% at 10 ⁻⁸ mg / ml, and the proliferative activity gradually decreased with increasing concentration. Proliferation activity of EtOAc was shown to 10 ^-7 mg / ml~10 ^-4 mg / ml in 114% showed a growth activity of ^{~120% 10 -3 mg / ml~10 -1} mg / ml 108% growth activity in It was. BuOH fraction also decreased with increasing concentration, which was similar to that of EtOAc fraction and showed 104 ~ 110% of proliferative activity at 10 ^-8 mg / ml ~ 10 ^-1 mg / ml. Osteocell enzyme activity of the horamified solvent fractions increased gradually from 10 ^-8 mg / ml to EtOAc fractions ranging from 112% to 118% up to 10 ^-2 mg / ml and from 140% at 10 ^-1 mg / ml. Showed the highest value. BuOH fractions, but also increased enzyme activity in proportion to the concentration increase showed a ^{10 -8 mg / ml~10 -2 mg /} ml 89% less active in ~92% of the 101% of the activity at 10 ^-1 mg / ml Seemed. The enzyme activity was proportional to the concentration when the Horam extract was fractionated and the activity of the EtOAc fraction was high (FIG. 2).

1.2) Isolation of Active Ingredients and Selection of Active Fractions

The EtOAc fraction, which had high activity as a solvent fraction, was fractionated by primary column chromatography (FIG. 3). Cell proliferation of Saos2 cells, a human-like osteoblastic cell line, was measured by MTT analysis at 10 ^-1 mg / ml, and the fractions 2 and 3 increased cell proliferation more than control. Cell proliferation rates were 206.9% and 228.2%, respectively. Fractions 4-7 showed higher cell proliferation rate than control, showing 158.9-178.4% proliferation rate, and other solid fractions showed relatively higher cell proliferation rate than control. Considered to be excellent. Alkaline phosphatase (ALP) activity was measured by applying Hosamu EtOAc extract, solid fraction to Saos2 cells at 10 ^-1 mg / ml concentration, and ALP activity of fractions 1-3 and 11-14 was relatively higher than that of the control. It was determined to be 124.8% to 110.2%. Fractions 4 and 10 showed similar ALP activity to the control, the remaining fractions showed lower ALP activity than the control, and fraction 8 showed the lowest ALP activity, which was 36.5%. As a result of primary chromatography, fractions having high proliferative capacity and ALP activity were separated by secondary column chromatography (FIG. 4). As a result of the second fractionation of the horamified primary chromatography fraction and MTT analysis, the cell proliferation ability of Saos2 cells, a human-like osteoblastic cell line, was found to be over 100% of the control, resulting in no cytotoxicity. In fraction 1, the proliferative capacity was high as 148% of control, fractions 6 to 10, fractions 12 to 15 were 105% to 125%, and fraction 19 was the highest at 153%. As a result of the second fractionation of the first homogenized fraction and the measurement of Alkaline phosphatase (ALP) activity, the fractions of 149% in fraction 5 and 138% in fraction 13 were high. Fractions were selected and combined. The separated fractions were subjected to 2-D TLC, purified active spots, and then characterized.

Example 2 Isolation and Structural Identification of Honed Bone Substrates Fortifying Active Materials

Compounds of one lignan and one alkaloid were isolated and identified according to the ALP activity-guided fraction.

2.1) Structure of Compound 1

Spectral properties of compound 1: ¹ H-NMR (400 MHz, pyridine- d ₅ , δ) 7.59 (1H, d, J = 8.5 Hz, H-5 '), 7.15 (1H, d, J = 2 Hz, H-2'), 7.04 (1H, dd, J = 8.5, 2 Hz, H -6 '), 6.91 (1H, d, J = 8.1 Hz, H-5), 6.90 (1H, d, J = 1.7 Hz, H-2), 6.86 (1H, dd, J = 8.1, 1.7 Hz, H-6), 5.66 (1H, d, J = 7.2 Hz, H-1``), 4.30 (1H, dd, J = 9.2, 8.4 Hz, H-9a), 4.16 (1H, dd, J = 8.4 , 7.6 Hz, H-9b), 3,79 (3H, s), 3.74 (3H, s), 3.73 (3H, s), 3.63 (1H, d, J = 13.2 Hz, H-7'a), 3.31 (1H, d, J = 13.2 Hz, H-7'b), 2.73 (1H, m, H-8) <sup>13</sup> C-NMR (100 MHz, pyridine- d <sub>5,</sub> δ) 178.67 (C-9 ') , 149.37 (C-3), 149.37 (C-3 '), 147.92 (C-4), 146.50 (C-4'), 131.84 (C-1 '), 129.71 (C-1), 122.80 (C- 6 '), 120.66 (C-6), 115.44 (C-5'), 114.59 (C-2 '), 112.73 (C-2), 112.10 (C-5), 101.74 (C-1''), 78.34 (C-5``), 78.00 (C-3 ''), 76.13 (C-8 '), 74.29 (C-2''), 70.61 (C-4''), 70.42 (C-9) , 61.73 (C-6 &quot;), 55.48 (OCH ₃ ), 55.39 (OCH ₃ ), 55.35 (OCH ₃ ), 43.61 (C-8), 41.09 (C-7 '), 31.31 (C-7).

Compound 1 has δ7.59 (1H, d), δ7.15 (1H, d), δ7.04 (1H, dd), δ6.91 (1H, d), δ6.90 (1H) in the ¹ H-NMR spectrum. Six olefinic methine protons were observed from the signals of, d) and δ6.86 (1H, dd), and it was estimated that they were derived from the benzene ring by chemical shift values. From signals of δ 4.30 (1H, dd) and δ 4.16 (1H, dd), one oxygenated methylene proton was present, and δ3.79 (3H, s), δ3.74 (3H, s) And three oxygenated methyl protons at δ3.73 (3H, s). In addition, one methine proton was identified at δ 43.61 (1 H, m), and δ 3.63 (1 H, d) and δ 3.31 (1 H, d), δ 3.24 (1 H, dd) and δ 2.94 (1 H). and dd), one methylene proton could be observed. At δ5.66 (1H, d), a number of methine protons and methylene protons were observed between δ4.2 and δ3.0 and the signals shown as anomeric protons, indicating that a single sugar was present. ^A total of 27 carbons were observed in the ¹³ C-NMR spectrum, and one esteric carbon signal was observed at δ178.67. At δ149.37 (× 2), δ147.92 and δ146.50, olefinic quaternary carbon signals with four oxygens were observed, and two olefinic quaternary carbon signals were observed at δ131.84 and δ129.71. Six olefinic methine carbon signals were identified at δ122.80, δ120.66, δ115.44, δ114.59, δ112.73 and δ112.10. As a result, it was found that two benzene rings exist. In δ76.13, oxygenated quaternary carbon signal was identified, and in δ71.32, one oxygenated methylene carbon signal was identified, and in δ55.48, δ55.39 and δ55.35, three oxygenated methyl carbon signals existed. Could. In δ43.61, δ41.09 and δ31.31, one methine carbon and two methylene carbon signals could be observed, and the sugar signal was found to be glucose by literature comparison. As a result, it was estimated that Compound 1 was bound to one molecule of glucose and three methoxy groups by a lignan compound bonded to two molecules of phenyl propanoid. 2D-NMR was measured to determine the more accurate structure and location of sugar and methoxy groups. ^A series of substructures could be estimated from the ¹ H- ¹ H COSY spectrum, and the exact assignment of each carbon and hydrogen was based on the HSQC spectrum. In the HMBC spectrum, a correlation was confirmed between the signal of δ 146.50 (C-4 ') and the anomeric proton signal of δ5.66 (1H, d), indicating that the sugar was bound to C-4'. Signals of 37 (x2, C-3, 3 ') and δ 147.92 (C-4) are δ 3.79 (3H, s), δ 3.74 (3H, s) and δ 3.73 (3H, s), respectively. By correlation with the signal of), it was confirmed that the methoxy group is bound to C-3, 3 'and 4. Based on this, compound 1 was identified as trakiloside which is a kind of lignan compound.

2.2) Structure of Compound 2

Spectral properties of compound 2: ¹ H-NMR (400 MHz, CD ₃ OD, δ) 7.41 (1H, d, J = 16.2 Hz, H-7 '), 7.38 (1H, s, H-4), 7.33 (2H, d, J = 7.8 Hz, H-2', 6 '), 7.19 (1H, d, J = 8.8 Hz, H-7), 7.01 (1H, s, H-2), 6.93 (1H, d, J = 8.8 Hz, H-6), 6.74 (2H, d, J = 7.8 Hz, H-3 ', 5'), 6.35 (1H, d, J = 16.2 Hz, H-8 '), 4.87 (1H, d, J = 7.6 Hz, H-1''), 3, 52 (2H, br s, H-9), 2.90 (2H, br s, H-8) ¹³ C-NMR (100 MHz, CD ₃ OD _, δ) 169.05 (C-9 '), 160.18 (C-4 '), 152.60 (C-5), 141.61 (C-7'), 134.25 (C-7a), 130.44 (C-2 ', 6'), 128.85 (C-1 '), 127.47 (C-3a) , 124.45 (C-2), 118.32 (C-8 '), 116.56 (C-3', 5 '), 114.13 (C-6), 113.11 (C-3), 112.45 (C-7), 106.59 ( C-4), 103.97 (C-1``), 77.96 (C-5 ''), 77.92 (C-3 ''), 75.05 (C-2 ''), 71.55 (C-4 ''), 62.63 (C-6``), 41.42 (C-9), 26.39 (C-8).

Compound 2 is As a result of ninhydrin color development, a pink spot was confirmed to be an alkaloid compound containing nitrogen, and in the ¹ H-NMR spectrum, the signal of δ 7.41 (1H, d) and δ6.35 (1H, which is coupled with 16.2 Hz) From the signal of d), two olefinic methine protons with trans binding were identified, δ7.38 (1H, s), δ7.33 (2H, d), δ7.19 (1H, d), δ6.93 Seven olefinic methine proton signals were identified at (1H, d) and δ6.74 (2H, d), confirming the presence of a benzene ring. In addition, one olefinic methine proton signal was confirmed at 7.01 (1H, s), indicating that a double bond other than the benzene ring was present. Two methylene proton signals were observed at δ41.42 (2H, br s) and δ26.39 (2H, br s), and signals seen as anomeric protons at δ4.87 (1H, d) and δ4.0-δ3 Between .0, multiple methine and methylene protons were observed, confirming the presence of one molecule of sugar. ^A total of 25 carbons were observed in the ¹³ C-NMR spectrum, and one olefinic quaternary carbon with oxygen was identified from the signal of δ 152.60, and the olefinic quaternary carbon signal and δ 127 which appeared to be combined with nitrogen at δ 134.25. At .47, one olefinic quaternary carbon signal was observed. In addition, one olefinic methine and one olefinic quaternaty carbon signal was identified at δ124.45 and δ113.11, respectively, and methylene carbon signal was observed at δ41.42 and methylene carbon at δ26.37. As a result of comparing literature, it was confirmed that Compound 2 was a compound having one molecule of serotonin. In addition to the serotonin structure, one ketone was found at δ 169.05, oxygen bound olefinic quaternary carbon at δ160.18 and olefinic quaternary carbon signals at δ128.85, δ130.44 (× 2) and δ116 Four olefinic methine carbon signals were identified at .56 (× 2). From the chemical shift value, it can be seen that one benzene ring exists, and in addition to the benzene ring, a pair of double bonds exist from signals of δ 141.61 and δ 118.32. In addition, the sugar signal was found to be glucose by literature comparison. Thus, it was estimated that Compound 2 is a compound in which a coumaroyl group and a sugar are bonded to one molecule of serotonin. 2D-NMR was measured to determine the more accurate structure and the location of the coumaloyl group and sugar. ¹ H- ¹ H COSY were able to estimate the correct part structure from the spectrum, it was possible to assign each of the right of the carbon and hydrogen based on a HSQC spectrum. In the HMBC spectrum, a correlation was confirmed between the signal of δ 169.05 (C-9 ') and the methylene proton signal of δ 3.52 (H-9), resulting in the coomaloyl group being bound to the terminal nitrogen present in C-9 of serotonin. The correlation was confirmed between the signal of δ 152.60 (C-5) and the anomeric proton signal of δ 4.87 (1H, d), indicating that the sugar was bound to C-5. In total, compound 2 was identified as N- (p-coumaroyl) serotonin mono-β-D-glucopyranoside. In the present invention, the identification of these compounds is of great significance as a method of extracting, separating, purifying, and identifying the bone-reinforcing core ingredients of homing, as well as using them as standards in the production process of products. The homing bone substrate strengthening active ingredient of the present invention will be utilized for a variety of products related to bone matrix strengthening, such as food materials, health food materials, pharmaceuticals, cosmetics.

Example 3: Construction of Bone Substrates Fortifying Active Osteocytic Lineage Assay

The sample used domestic homing. The effect of the sample extract on the degree of cell differentiation and cell-based bone matrix strengthening activity was compared using human-like osteoblast cells (MG-63 cells / Saos-2 cells). Influence test on cell differentiation of sample extract was adjusted to 2 × 10 ⁴ cells / well, and the cells were divided into 96-well plates with each concentration sample and incubated for 2 days. The test group cultured only with the culture medium was used as a control group, and the sample group was applied by concentration (1 × 10 ^-1 to 10 ^-8 mg / ml), and the positive control group was NaF (Sigma Co., USA) and 1, 25 (OH) ₂ D ₃ was used. On the day of the analysis, 20 μl of MTT (3- [4,5-dimethyl-thiazol-2-yl] -2,5-diphenyltetrazolium bromide, Sigma Co., USA) solution (final concentration 0.5 mg / ml) was applied per well of 20 μl. After incubation for 4 hours, the culture solution was removed, 150 μl of dimethylsulfoxide (DMSO) was added, and the MTT formazan was dissolved for 5 minutes. After 5 minutes, the absorbance (540 nm, 25 ° C.) was measured by ELISA reader and compared with the control% ratio. Bone matrix strengthening activity of the sample extract as a positive control group Alkaline phosphatase (ALP) activity as a measure. The activity of Alkaline phosphatase (ALP), which is used as an indicator of osteoblast activity in human MG-63 cells and Saos-2 cells, was measured. Cell cultured only with culture medium was used as a positive control. ALP activity was measured to promote the decomposition of colorless p-nitrophenylphosphate to yellow p-nitrophenol and phosphate. First, as in the MTT experiment, Saos-2 cells were adjusted to 2 × 10 ⁴ cells / well, divided into 96 well plates with samples for each concentration, and cultured for 48 hours. The cell group using only the culture medium was used as a control group, and the sample group was applied by concentration (1 × 10 ⁻¹ to 10 ⁻⁸ mg / ml), and soybean was used as a positive control group. After incubation for 48 hours, 5 μl / well of 10% Triton x-100 / in PBS (final concentration) and 300 μl / well of 1% Triton x-100 / in PBS (final concentration) were dissolved on ice, followed by sonication (15). sec) and centrifuged (14,000 rpm, 20 min, 4 ° C). After centrifugation, the supernatant was added to 4 µl 96-well plate and 200 µl of ALP test kit reagent (Thermo Trace kinetic method kit) was added, followed by reaction for 1 minute using a microplate reader (405 nm), followed by measurement for 2 minutes. Comparison was made with the control% ratio.

Example 4 Construction of Bone Inhibition Test Method

The bone destruction inhibition test of the sample extract was divided into the inhibitory activity of precursor which is the early differentiation inhibitory activity of osteoclast formation and the differentiation inhibition of multinucleate after osteoclast formation. Osteoclast precursor cells (precursor cells) were dispensed into 96-well plates at 6 × 10 ⁴ cells / well and incubated in α-MEM with M-CSF added for 3 days. Differentiation was tested by administering a sample to progenitor cells compared to the control and measured as inhibitory activity against the differentiation of osteoclast precursors. Multinucleate differentiation inhibition test after osteoclast formation induced the differentiation of osteoclasts by adding RANKL, and the samples were added together. After adding RANKL and samples, the cells were cultured for 6 days, and the osteoclasts were fixed and stained using the TRAP stain kit. The medium was changed every 3 days in culture. The cells with the number of nuclei in the osteoclasts stained under the microscope were counted. The effect on the osteoclast marker enzyme TRAP activity was dispensed into 96-well plate of osteoclast progenitor cells 6 × 10 ⁴ cells / well and incubated in α-MEM added M-CSF for 3 days. After that, RANKL was added to induce differentiation of osteoclasts, and samples were added together. After adding RANKL and sample, the cells were incubated for 6 days. After incubation, the cells were treated with 0.1% Triton X-100 / saline, and a certain amount of the cell treatment solution was used for measuring TRAP enzyme activity. In the presence of 100 mM p-nitrophenyl phosphate (PNPP) and tartrate solution as substrates, 0.1M After reacting with sodium citrate buffer (pH 4.9) for 10 minutes at 37 ℃, the amount of p-nitrophenol (PNP) released from the substrate PNPP was colorimetrically determined at 405 nm. A certain amount of cell treatment was used to quantify the protein using the Lowry method and to calculate the TRAP activity (activity: cleaved μmol substrate / min / mg protein).

Example 5: Cell Line Activity of Hormone Bone Substrate Enhancing Composition

5.1) Bone Substrates Enhancement Activity of Osteoblasts

The cell proliferation of Saos-2 cells and MG-63 cells, which are human-like osteoblasts, were compared using 1.25 (OH) ₂ D ₃ and NaF as positive controls (FIG. 5). In the case of Saos-2 cells, 1,25 (OH) ₂ D ₃ has exhibited a 90% control at 10 ^-8 mg / ml concentration of NaF is the control 97% of control at 10 ^-8 mg / ml concentration of indicator substance Cell proliferation was increased in proportion to the concentration of (NaF), and domestic proliferation was more stable than the positive control group from 1 × 10 ^-8 mg / ml to 100% proliferation. In the case of MG-63 cells, 1,25 (OH) ₂ D ₃ decreased with increasing concentration, NaF showed more than 100% cell proliferation at all concentrations, and domestic homogenization was the same as that of Saos-2 cells. The cell proliferation was superior to the positive control with 100% proliferation. For ALP activity (FIG. 6), 1,25 (OH) ₂ D ₃ showed an increase of 131% to 155% ALP activity of the control at a concentration of 1 × 10 ^{−4 to} 1 × 10 ^-7 mg / ml, and 1 × 10 A high ALP activity increase of 168% was shown at the concentration of ^-8 mg / ml. NaF showed an increase in activity of 115% to 145% of the control at a concentration of 1 × 10 ^{-5 to} 1 × 10 ^-8 mg / ml and a high increase of 155% of the control at a concentration of 1 × 10 ^-4 mg / ml. Seemed. Domestic call ramhwa by the ALP activity increased from ^{1 × 10 -8 ~1 × 10 -6} mg / ml concentration showed a maximum value to 127% of that of the control at 1 × 10 ^-6 mg / ml concentration was similar to the positive control .

5.2) osteoclast inhibitory activity

Hormone extract showed 24.1%, 79.5% of precursor cell inhibitory activity at 1㎍ / ml, 25㎍ / ml and 39.5%, 99.8% of multimucleate cell differentiation inhibitory activity. At concentrations of 50 µg / ml or more, osteoclast inhibitory activity was not observed due to cellular stress. The osteoclast inhibitory activity showed the highest activity at the concentration of 25 µg / ml. Hormone extract showed higher inhibitory activity on differentiation of multinucleate cells than the interaction of the soluble receptor activator of NF-κB ligand (RANKL) and OPNK (RANK-osteoprotegerin of osteocylast progenitor) in Osteoblast and stromal cell membranes (Fig. 7).

Example 6: Hormonal Bone Substrates Enhancement Compositions

Horamhwa is a domestic horamhwa cultivated and provided by [Uiseong Woohonghwain Farming Association]. , P, Mg, Casein phospho peptide, Vitamin D _3, etc.) was developed a homing bone matrix strengthening composition (Fig. 8). Hormonalization and bone matrix strengthening verification of the composition (hereafter sample extract) were scientifically validated using human-like cell lines and ovarian isolated animal assays.

Example 7: Bone Substrates Enhancement Physicochemical Properties of Horamophilic Extracts

Physicochemical properties were tested to indirectly confirm the maintenance and in vivo activity of homing bone matrix strengthening activity during processing. The effect on temperature was measured for bone matrix strengthening activity after treatment of the Horamophilic extract at 60 ℃, 80 ℃, 100 ℃ and the effect on heating time was measured after treatment at 100 ℃ reflecting the processing conditions. Bone matrix strengthening activity of the Horamyeo extract showed a differentiation degree of 107% to 116% at temperature changes and 89% to 105% of ALP activity was also maintained (Fig. 9). The effect on the heating time of the eosinophilic extract was maintained over 100% of the degree of differentiation during heating, and the ALP activity was 126-131% up to 6 minutes, but rapidly decreased from 8 minutes to 98%. The heating time was lowered to 89% or less when exceeding 10 minutes (FIG. 10). Osteoblast proliferation and ALP enzyme activity according to pH change of the Horamophilic extract in acidic, neutral and basic conditions were maintained at 118-138% proliferative activity and 100% or more ALP activity according to pH change (FIG. 11). . It has been confirmed that the stabilized bone matrix strengthening active ingredient is stable to temperature change and pH change and maintains bone matrix strengthening activity during processing or in the body.

Example 8: Construction of Bone Substrates Enhancing Active Animal System Assay

8.1) Inhibitory Effect of Osteoporosis Using Cockerel

Freshly hatched broiler male spawning chicks (breed: Hy-Line Brown) were used to investigate the effects of horus on the organ and bone development of young animals. A total of 300 male chicks (approximately 34 g in weight) were bred and reared at 7 days of age, four replicates were selected per treatment so that there was no difference in average weight among the treatment groups. . During the experiment, the experimental feed and water were supplied to ad libitum , and the temperature of the juvenile chamber was decreased by 2 ° C every week from the initial 34 ° C. The lighting was carried out 24 hours a day, and other specification management was carried out in accordance with laboratory practices. For dietary protein source, soy protein concentrate (SPC) with very low total isoflavone content of 41 μg / g protein was used. In the basic feed, all nutrients except calcium were formulated according to NRC specification standard. Crude protein was 19% and ME (metabolizable energy) was 3,100 kcal / kg. The experimental period was 3 weeks in total. To evaluate the effects of horamization on bones, 30% of the total calcium requirement was added to the basal diet to induce osteoporosis and the amount of available P was adequate. It was added to prevent phosphorus from acting as a limiting factor. Experimental feed was replaced with a part of the cellulose content (3% of total) added to the basic feed as a sample extract, CaCO ₃ The calcium content of the basic feed and the extract was compared and corrected. Until the 2 weeks of preliminary specification period, a single commercial feed was fed, and from 3 weeks of age, a homogenized experimental feed was provided.

8.2) Suppression of Osteoporosis in Ovariectomized Female Rats

Sprague-Dawley female rats (110 g), 6 weeks old, were divided into two groups: Sham, control group, and control group (S group, E group) for the treatment of osteoporosis. Bone matrix strengthening experiments were conducted. One week after the adjustment period, ovarian extraction was performed, and 12 weeks later, the cervical spine was dislocated and blood collected from the eye. Serum Alkaline phosphatase activity was analyzed by autoanalyzer (ADVIA 1650, Bayer, Japan) using Buffer / Enzyme Reagents and ALP reagents (Bayer, USA) consisting of ALT co-enzyme. Osteocalcin was a competitive method. The analysis was performed using an autoanalyzer (ADVIA 1650, Bayer, Japan). Results The serum ALP of the composition containing the hormone-enhanced bone matrix enhanced component was 8,441 IU / L in the cockerel compared to 8,314.4 IU / L in the control group and was similar to that of the sham group, which is a normal experimental animal of ovariectomy ( 12). Serum calcium and phosphorus ratios were also higher than that of controls and similar to sham.

Example 9 Supercritical Extraction Method of Hormonal Bone Substrates Fortified Active Ingredients

In addition to solvent extraction, supercritical extraction was performed as another extraction method for the bone matrix strengthening active ingredient. The sample was placed in a cylinder as a raw material and then circulated and extracted with supercritical carbon dioxide. The supercritical fluid was sequentially rotated to 2500 L CO ₂ at 50 ° C. and 5000 psi to obtain seven extraction fractions by weight. Fatty acid content of each fraction as a promoter of bone cell activation and calcium absorption was analyzed by gas chromatography and detected by a FID detector (FIG. 13). Linoleic acid was the highest with more than 80%, oleic acid 10%, other palmitic acid 5% -6%, stearic acid 1% -2%, etc. The possibility was confirmed (FIG. 14). The content of phospholipid, which is a substrate for osteoblast activation, was analyzed by inductively coupled plasma atomic emission spectroscopy (ICP) using inductively coupled plasma-atomic emission spectrophotometer (ICP). The calibration curve using four points was prepared and measured (FIG. 15). Phospholipids were not detected in addition to neutral lipids, but 620 mg / g of phospholipids were detected in the extracted foil. Phospholipids have the potential to activate intermediate substrates during bone matrix strengthening, so phospholipids can be recovered from the extract foil or the extract foil can be used as a solid. When processing bone matrix strengthening active ingredients, we can utilize low-molecular core ingredients with high activity in solvent extraction and diversify the extraction method to obtain products such as linolenic acid and phospholipids by supercritical extraction to construct products.

Example 10 Oxidative Stability of Horamophilic Extracts Containing Bone Matrix Enhancing Active Ingredients

In the preparation of the Hohwa oil using the horamification, the sample was prepared only by simple pressing without the roasting process. BHA. BHT, PG, α-tocopherol, and TBHQ were used as antioxidants. The active extract extracted with 80% ethanol was homogenized overnight at room temperature, and then repeatedly filtered with whatman No. 2 and No. 40. The filtrate was concentrated under reduced pressure at 40 ℃. Accelerated storage test was performed to investigate the effects of various antioxidants during the automatic oxidation. Samples containing 0.02% of antioxidants were stored at 60 ° C. in a constant amount, and then aliquoted at regular intervals. The peroxide value was measured according to the method of A.O.C.S. In accordance with the method described in the Food and Drug Administration, 5 g of homogenized oil was taken in a 250 ml Erlenmeyer flask, and then dissolved by adding 100 ml of ethanol / diethyl ether (1: 2, v / v) solution, and phenolphthalein solution was used as an indicator. The acid value was determined by titration until the pale red color lasted 30 minutes with hydroxide ethanolic solution. The result showed that the TBHQ added 0.02% showed the slowest increase during the storage period and the antioxidant activity was 41% after 50 days of storage, which was the best antioxidant among the antioxidants used in the experiment. In addition, when TBHQ was mixed with α-tocopherol, the peroxide value of the control increased rapidly as the storage days increased, whereas the antioxidant treatment increased at a slow rate, showing an inhibition rate of 69% after 21 days of storage. TBHQ and tocopherol were effective for oxidative stabilization.

Example 11: Materialization utilizing technology

11.1) Dispersion of Horamophilic Extracts in Aqueous Solution

A dispersion composition in aqueous solution was developed as a basis for the development of a liquid processing material of hohwa containing the bone matrix strengthening component. Arabian gum, carrageenan, and sucrose fatty acid esters were used in combination from 0.1% to 0.7% as an emulsifier for dispersion in aqueous solution after preparation of 1% aqueous solution. In addition, the extract containing the hormonal strengthening component for the first time was applied optically to the Turbiscan and analyzed optically, and the particle dispersion, creaming, and sedimentation were calculated as the inverse of the back scattering with respect to the wavelength. The control group was divided into sedimentation and flotation as soon as shaking, and there was no dispersion of bone matrix-enhanced active material and dispersibility was shown in the lower layer when emulsifier was treated, but the floating state remained due to the increase of BS in the upper layer. When 0.7% sucrose fatty acid ester was added, variation occurred in the overall solution state and the BS was uniformly dispersed (FIG. 16).

11.2) Development of solid products such as powders and pills

Solid products such as powders and pills were developed using the homogenized active extracts (FIGS. 17 and 18). The washed samples were dried for 12 hours at 80 ° C and roasted at 180 ° C for 5 minutes to stabilize surface moisture and wax components. After cooling the air to room temperature and repeatedly pulverized more than 70 times with a roll mill, and re-crushed with a hammer mill to prepare a powder sample by 180mesh treatment. Rings are prepared by mixing dough, molding, coating, and packing. Mixing dough is kneaded twice with a roll mill, and then extruded into a shape of about 30 cm by extrusion process. The primary molding was carried out and the secondary molding was carried out through a ring converting machine to form a ring. Coating and packaging were dried after molding and packaged by coating the surface with honey or shellac.

Since the homogenized or the extract of the present invention can improve the physiological diseases that cause osteoporosis, it is judged to be of sufficient value to be used as a material for medicines and health functional foods for improving bone diseases in adults in the future and is expected to be practical.

Figure 1 shows the results of the qualitative analysis of the solvent extraction, fractionation process and the material profile of the homogenization.

2 is a measurement of osteoblast proliferation and enzyme activity for each solvent fraction.

Figure 3 shows the partitioning by primary column chromatography on the EtOAc fraction. Fractions 2 and 3 more than doubled the cell proliferation than the control.

Figure 4 shows the separation of the separation by secondary column chromatography on the fraction of high proliferation capacity and ALP activity as a result of the primary chromatography. In total, there was no cytotoxicity as it exhibited more than 100% activity of the control.

FIG. 5 compared the cell proliferation of Saos-2 cells and MG-63 cells, which are human-like osteoblasts, using 1.25 (OH) ₂ D ₃ and NaF as positive controls.

Figure 6 compared the ALP activity of Saos-2 cells and MG-63 cells, which are human-like osteoblasts, using 1.25 (OH) ₂ D ₃ and NaF as positive controls.

Figure 7 shows the synthesis of bone substrate strengthening and bone absorption (osteogol) characteristics of the Hohwa extract.

FIG. 8 developed a homogenized bone matrix strengthening composition based on a homogenized extract.

Figure 9 shows the bone matrix strengthening activity of the horamyeo extract according to the heating temperature. The degree of differentiation of 107% to 116% was observed at the temperature change of 60-100 ° C, and the activity of ALP activity was 89% to 105%.

Figure 10 shows the bone substrate strengthening activity of the horamyeo extract with heating time. The eosinophilic extract maintained more than 100% of the degree of differentiation even during heating, and the ALP activity was stable at 126% ~ 131% up to 6 minutes.

Figure 11 shows the bone matrix strengthening activity of the Horamyeo extract according to pH. According to the pH change, the growth activity was maintained at 118 ~ 138%, ALP activity 130% or more.

Figure 12 shows the animal-based serum ALP content and the content of Ca, P of the composition comprising the Horamophilic extract and the Hormone bone matrix strengthening component.

Figure 13 shows the fatty acid content of each fraction as a promoter for bone cell activation and calcium absorption, and was analyzed by gas chromatography and detected by a FID detector.

14 was analyzed by gas chromatography for the degree of recovery of linoleic acid.

15 was analyzed by inductively coupled plasma atomic emission spectroscopy using an inductively coupled plasma-atomic emission spectrophotometer (ICP).

FIG. 16 is most stably dispersed when 0.7% sucrose fatty acid ester is used as a result of dispersion using an emulsifier in an aqueous solution of a homogenized extract.

Figure 17 shows the process of developing a solid product using the homogenized active extract.

Figure 18 was developed a solid product according to the formulation of health functional materials, such as powder, pills, using a horamyeo active extract.

Claims (9)

Trad kilo side (tracheloside) or N- (p - one to Kumar) serotonin mono - β -D- gluconic containing nose Llano side (N- (p -coumaroyl) serotonin mono- β -D-glucopyranoside) as an active ingredient Further, at least one selected from the group consisting of fresh green grass extract, ganoderma lucidum extract, prickly pear extract, soybean extract, pomegranate extract, burdock extract, broiler extract, Ca, P, Mg, casein phospho peptide (CPP) and vitamin D ₃ Health food composition for bolstering the bone matrix to activate the osteoblast system containing, inhibiting the osteoclast system. delete delete delete Containing a healthy food composition for strengthening bone matrix according to claim 1, a liquid preparation for a healthy food for strengthening bone matrix. The liquid preparation according to claim 5, wherein sucrose fatty acid ester is used to improve the dispersibility of the aqueous solution. A solid preparation for bone food reinforcement health foods containing the health food composition for bone matrix reinforcement according to claim 1. delete delete

Images