KR20160096357A - Composition for preventing or improving bone-related disease comprising polysaccharide purified from Styela plicata extract as effective component - Google Patents

Abstract

The present invention relates to a health food composition and a pharmaceutical composition which contain refined polysaccharide derived from Styela plicata as an active ingredient to prevent or alleviate a bone-related disease, wherein the refined polysaccharide is isolated by adding water and protease to Styela plicata powder, making an extract, and performing ion exchange chromatography. The health food composition and the pharmaceutical composition have enhanced alkaline phosphatase activity and bone calcification ability.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or ameliorating a bone-related disease comprising an osteoporotic polysaccharide purified product as an active ingredient.

The present invention relates to a method for the treatment of Styela plicata powder and extracting it with water and a protein hydrolyzing enzyme, and using the ion exchange chromatography as an active ingredient, a health functional food composition and a pharmaceutical composition for preventing or ameliorating a bone related disease ≪ / RTI >

Bone tissue is composed of various kinds of cells such as extracellular substance such as collagen and glycoprotein, osteoblast, osteoclast, and osteocyte. In particular, bone resorption by osteoclasts and new bone matrix formation and mineralization by osteoblasts are repeated metabolic processes, and osteoblastic activity is greater than osteoclast activity. The bone resurfacing is the process of removing the old bone after growth and replacing it with a new bone. It is the process of removing hormones such as parathyroid hormone (PTH), calcitonin and estrogen, and bone tissue such as insulin-like growth factor I It regulates the activity balance of osteoblast and osteoclast through cytokines such as secreted various growth factors and tumor necrosis factor-alpha (TNF-α), and maintains homeostasis. Continuous linkage between bone formation and bone resorption is controlled by various factors such as systemic hormones and local regulators such as cytokines. NOS (nitric oxide synthase), an enzyme that produces NO, 17 (interleukin-17), and TNF-alpha (tumor necrosis factor-alpha).

Korea has already entered an aging society and has an aged society that is over 65 years old with an age of over 14%. Osteoporosis, a bone metabolic disease that is becoming the biggest problem in the aging society, is a disease that can cause bone fracture easily due to a decrease in the bone volume in the unit volume without significant change in the chemical composition of the bone. But bone resorption by osteoclasts is increased, resulting in rapid bone loss. Especially, it has bone metabolic diseases such as decrease in bone mineral content of about 20% within 10 years after menopause. Treatment of osteoporosis is known as female hormone replacement therapy, selective estrogen receptor modulator (SERM), bisphosphonate, parathyroid hormone, and calcitonin. Currently, estrogen or phytoestrogen, which is present in nature and has weak estrogenic activity, is very useful for the prevention and treatment of postmenopausal osteoporosis.

Osteoblasts originate from the mesenchymal stem cells of the bone marrow and are involved in bone formation and calcification that can differentiate into contracts, myocytes, and adipocytes. Alkaline phsophatase (ALP), which is a glycoprotein, is present in the cell membrane. It has been found to be optimal in substrate specificity and basic pH, and high in the outer membrane of the cell and in the matrix vesicles of the calcified tissue. It is known as a marker of osteocyte differentiation that acts as a regulator of cell division and differentiation.

Among the various marine organisms, Styela (also known as white myrtle or wrinkle muddy) plicata ) is a marine creature belonging to microalgae, seaweed, and lateral seaweed, which is a kind of rhizomes. Its tip is attached to a rock, attached to shells and rocks, and grows like a creature of hermaphrodite. The creature is known as the midduk and the ukiyoe. It is somewhat rounded compared to midduck, has no tail, has a thick, somewhat pale color on the surface of the shell, has a unique flavor and taste, and is widely used in foods such as steamed or miso soup. In Oman, heparin and dermatan sulfate have been shown to have anti-coagulant effects, and the presence of an octapeptide called plicatamide as an antimicrobial peptide has been reported, hemocyte) showed cytotoxicity against human K-562 tumor cell line. Studies have also been reported on the antioxidant activity and the in vitro cytotoxic effect of the Omani extract.

Korean Patent No. 1320974 discloses a composition for prevention and treatment of bone diseases containing an extract of Shin as an active ingredient and Korean Patent No. 1306876 discloses a composition for preventing or treating bone diseases But is different from a health functional food composition for preventing or ameliorating a bone related disease containing the purified omal polysaccharide of the present invention as an active ingredient.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and an object of the present invention is to provide a method of extracting omalis from alkaline phosphatase (alkaline phosphatase), which solves the problems of human toxicity, low extraction yield and high cost, phosphatase activity and osteoporotic polysaccharide improved in bone formation ability as an active ingredient as an active ingredient, and to provide a health functional food composition and a pharmaceutical composition for preventing or improving bone related diseases.

In order to solve the above-mentioned problems, the present invention provides a styela plicata powder and extracting it with water and a protein hydrolyzing enzyme, and using the purified omal polysaccharide separated by ion exchange chromatography as an active ingredient, to provide a health functional food composition for preventing or ameliorating a bone related disease do.

In addition, the present invention relates to a method for the treatment of Styela The present invention provides a pharmaceutical composition for preventing or treating a bone-related disease containing, as an active ingredient, a purified omal polysaccharide obtained by adding water and a protein hydrolyzing enzyme to plicata powder and extracting it using ion exchange chromatography.

The extract of Oman polysaccharide of the present invention has a higher production of nitric oxide and cytokine and enhances the expression level of immune-related genes compared to the extract of Omani, and does not exhibit cytotoxicity to osteoblast cells but alkaline phosphatase activity And to improve the ability to form bone calcification to prevent or ameliorate bone related diseases, and the like.

Figure 1 shows that the crude polysaccharide is packed in a column using DEAE-sepharose CL-6B as a filler, equilibrated with 50 mM sodium phosphate buffer (pH 6.0), and gradient of 0-1 M NaCl to give a flow rate of 1 mL / min (Fraction No. 25-31) and SPF-II (fraction No. 25-31) eluted with NaCl solution after being adsorbed to non-adsorbed fraction (SPF-I, fraction No. 16-21) eluted with distilled water and SPF -III (fraction No. 36-41) and uronic acid content. F1: SPF-I, F2: SPF-II, F3: SPF-III
Fig. 2 is a photograph of a gel and balm-type finished product for prevention and improvement of arthritic diseases containing osmotic polysaccharide purified water.
FIG. 3 is a graph comparing the amounts of nitric oxide (NO) produced in the omnidisole polysaccharide extract (SP) and purified polysaccharide (SPF-I, SPF-II and SPF-III).
FIG. 4 is a graph comparing cytokine TNF-.alpha. Production of Oman saponin extract (SP) and purified polysaccharide (SPF-I, SPF-II and SPF-III).
FIG. 5 is a graph showing the results of a total RNA extraction after culturing Oman polysaccharide purified with a macrophage cell line at a concentration of 0.05-0.5 / / mL, synthesizing cDNA with a reverse transcriptase, and expressing mRNA by PCR using a primer The results are shown in Fig.
FIG. 6 is a graph comparing the proliferation rates of osteoblast cells according to the treatments of osmotic polysaccharide tablets (SPF-I, SPF-II and SPF-III).
7 is a graph comparing the activities of alkaline phosphatase (ALP) in Osteoblastic cells with Omani extracts (SP) and purified polysaccharides (SPF-Ⅰ, SPF-Ⅱ and SPF-Ⅲ).
FIG. 8 is a photograph showing a comparison of the ability of osteonecrosis polysaccharide extract (SP) and the polysaccharide purified product (SPF-I, SPF-II and SPF-III)
Con (Control) in FIGS. 3 to 8: Control (untreated).
FIG. 9 is a graph comparing pH stability of the gel and ointment-type finished product for prevention and improvement of osteoporosis-associated polysaccharide-containing joint disease according to storage temperature and period.

According to an aspect of the invention, the invention Oman snout (Styela plicata powder and extracting it with water and a protein hydrolyzing enzyme, and using the purified omal polysaccharide separated by ion exchange chromatography as an active ingredient, to provide a health functional food composition for preventing or ameliorating a bone related disease do.

In the health functional food composition for preventing or ameliorating the bone related disease of the present invention, the protein hydrolyzing enzyme may be a protease such as neutrase, but is not limited thereto.

In the health functional food composition for preventing or ameliorating a bone related disease of the present invention, the purified omal polysaccharide is a non-adsorbed fraction eluted with distilled water when eluted with a 0 to 1M salt concentration gradient using ion exchange chromatography . The non-adsorbed fraction, SPF-I ( Styela plicata fraction-I) was higher than that of SPF-Ⅱ and SPF-Ⅲ, which were eluted by NaCl solution, and the activity of alkaline phosphatase was higher than that of SPF-Ⅲ.

In addition, in the health functional food composition for preventing or ameliorating the bone related disease of the present invention, the extraction can be preferably performed at 50 to 70 ° C for 12 to 18 hours, more preferably at 60 ° C for 15 hours But is not limited thereto.

In the health functional food composition for preventing or ameliorating a bone related disease according to the present invention, the bone related disease is selected from the group consisting of osteoporosis, osteoarthritis, degenerative arthritis, knee arthritis, arthropathy, systemic lupus erythematosus, spinal arthritis, Acute rheumatic fever, gout, cartilage calcification, periodontal disease due to alveolar bone destruction, inflammatory alveolar bone disorder, inflammatory bone resorption disease, ulcerative colitis, ulcerative colitis, chronic myelogenous leukemia, myalgia, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, intestinal arthritis, rheumatoid arthritis, , Calcium hydroxyapatite crystal precipitation disease, and lime disease. However, the present invention is not limited thereto.

The health functional food is not particularly limited as long as it can be ingested for prevention or improvement of bone related diseases.

When the purified omal polysaccharide of the present invention is used as a food additive, the purified product can be used as it is, or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the purified product of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, in the production of food or beverage. However, in the case of long-term consumption intended for health or hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the foods to which the purified water can be added include meat products, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen noodles, other noodles, dairy products including ice cakes, various soups, drinks, tea, , Alcoholic beverages and vitamin complexes, and includes all health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the purified water of the present invention.

In addition to the above, the purified product of the present invention may be used in various forms such as various nutrients, vitamins, electrolytes, flavors, colorants, salts of pectic acid, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, A carbonating agent used in a carbonated beverage, and the like. In addition, the tablets of the present invention may contain flesh for the production of natural fruit juice, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention also relates to a method for the treatment of Styela The present invention provides a pharmaceutical composition for preventing or treating a bone-related disease containing, as an active ingredient, a purified omal polysaccharide obtained by adding water and a protein hydrolyzing enzyme to plicata powder and extracting it using ion exchange chromatography.

In the pharmaceutical composition for preventing or treating bone related diseases of the present invention, the pharmaceutical composition may be prepared into a gel or balm type. The gel is prepared by adding deionized water, ethanol , Ointment, chamomile extract, methanol, camphor, aloe vera powder, carrot extract, Carbopol 940, triethanolamine, methylparaben, propylparaben and blue No. 1, But are not limited to, vaseline, shea butter, camphor, methanol, squalane, macadamia oil, avocado oil, jojoba oil, grape seed oil, evening primrose oil and coconut oil.

The pharmaceutical compositions containing the Oman polysaccharide tablets of the present invention may further comprise suitable carriers, excipients or diluents according to conventional methods. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions according to a conventional method have. More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, Can be prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of liquid formulations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition of the present invention may vary depending on the patient's age, sex, and body weight, but is generally administered in an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, can do. In addition, the dosage of the tablet can be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Since the purified product of the present invention has little toxicity and side effects, it can be safely used for prolonged use for preventive purposes.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

1. Experimental material

Styela plicata used in this experiment was supplied from the domestic fishery product maker located in Masan, Gyeongnam and was used for the experiment. (OF-22, JEIO TEC, Daejeon, Korea), followed by pulverization with a pulverizer (J-NCM, Jisico, Seoul, Korea) to obtain a 60 mesh standard netting (Chung Gye Sang Gong Sa., Korea) was used as an extractive sample while being kept in a cow below -20 ℃.

2. Omani  Polysaccharide Purified water  Produce

(1) Extraction of polysaccharides

The extraction of crude polysaccharide from Oman was carried out by adding 20 times of distilled water to 10 g of hot air-dried Omani crushed product and adding Nutraze (Novozymes, Bagsvaerd, Denmark) so that the protein concentration ratio to the substrate was 1/50 And the mixture was stirred and extracted at 60 DEG C and 150 rpm for 15 hours using a shaking incubator (BS-31, JEIO TEC, Daejeon, Korea). The extract was centrifuged (3,000 × g, 20 min, VS-6000CFN, Vision Scientific Co., Ltd., Bucheon, Korea), and the supernatant was added with trichloroacetic acid (TCA, Acros organics, Geel , Belgium) was added and allowed to stand at room temperature for 30 minutes, followed by centrifugation (3,000 × g, 20 minutes) to remove precipitated proteins. The supernatant from which the protein was removed was concentrated under reduced pressure (Model N-1N, Eyela Co., Tokyo, Japan), and 5-fold amount of ethanol was added to the concentrate. The mixture was stirred for 24 hours and centrifuged (3,000xg, 20 minutes) (FreeZone 2.5, Labconco Co., Kansas, MO, USA) and stored in a dark room at -70 ° C or lower while being used as a sample for separation and purification.

(2) Separation and purification of polysaccharides

The crude polysaccharide was dissolved in 50 mM sodium phosphate buffer (pH 6.0) (50 mg / mL) and centrifuged (3,000 × g, 20 min, VS-6000CFN, Vision scientific Co.) After removing the material, the column was packed into a column (2.5 × 30 cm) using DEAE-sepharose CL-6B (Amersham Bio-sciences Co., Piscataway, NJ, USA) as a filler, and 50 mM sodium phosphate buffer , And the concentration was adjusted to 0 to 1 M NaCl to obtain a concentration of 10 mL at a flow rate of 1 mL / min. Fractions SPF-II (fraction No. 25-31) and SPF-III (fraction No. 36), which were adsorbed to non-adsorbed fraction (SPF-I, fraction No. 16-21) eluted with distilled water and then eluted by NaCl solution, -41) and the results of analysis of uronic acid content are shown in Fig.

The fractions were collected by phenol-sulfuric acid method for total sugar and Cesaretti for uronic acid, and dialyzed and concentrated by distilled water, followed by lyophilization and storage at -70 ° C. Respectively.

3. Experimental Method

(1) Cell culture

RAW 264.7 (KCTC No.40071) cells used in the experiment were purchased from Korean Cell Line Bank (KTCC, Seoul, Korea). Cell culture was performed by adding 10% fetal bovine serum (Gibco BRL Co., Grand Island, NY, USA) and 1% penicillin-streptomycin (Gibco BRL Co.) using DMEM medium (Welgene, Daegu, Korea) Lt; / RTI > Cells were all cultured in a CO ₂ incubator (MCO-18AIC, SANYO Co., Sakata, Japan) adjusted to 5% at 37 ° C.

(2) Nitric oxide ( Nitric oxide , NO ) Production amount measurement

RAW 264.7 cells were added to 96-well plates at a concentration of 5 × 10 ⁴ cells / well and cultured for 24 hours. Each sample was treated with LPS (0.1 μg / mL, Sigma-Aldrich Co.) Time. After incubation, 50 μl of the supernatant was mixed with the same amount of grease (Sigma-Aldrich Co.) reagent and reacted for 10 minutes. Absorbance was measured at 540 nm using a microplate reader (UVM-340, ASYS Co.) The amount of the produced amount was calculated using the standard curve of the concentration of sodium nitrite (Sigma-Aldrich Co.).

(3) cytokines cytokine )

The crude polysaccharide and polysaccharide purified from Oman were extracted into 24-well plates of RAW 264.7 cells at a concentration of 5 × 10 ⁵ cells / well, cultured in an incubator for 24 hours, and then cultured in a culture supernatant The amounts of TNF (tumor necrosis factor) -α and IL-6 were measured by an ELISA kit (Pepro tech Inc., Rocky Hill, NJ, USA) according to the manufacturer's instructions. The contents of TNF-α and IL-6 were calculated using standard curves of TNF-α and IL-6 contained in the kit.

(4) RT - PCR

A sample and LPS (0.1 mg / mL) were treated with a RAW 264.7 at a concentration of 1.5 × 10 ⁶ cells / well and incubated for 6 hours. Cells were collected and centrifuged to remove the supernatant, (1 μg) was obtained by using RNeasy mini kit (Qiagen, Hilden, Germany). The cDNA was synthesized with Revertaid first strand cDNA synthesis kit (Thermo Scientific, Waltham, MA, USA) RT-PCR (Mastercyclernexus, Eppendorf AG, Hamburg, Germany) was performed. The primers used were iNOS (sense, 5'-CCCTTCCGAAGTTTCTGGCAGCAGC-3 '(SEQ ID NO: 1), antisense, 5'-GGCTGTCAGAGCCTCGTGGCTTTGG-3' (SEQ ID NO: 3), antisense, 5'-ATGCTCCTGCTTGAGTATGT-3 '(SEQ ID NO: 4), TNF- 5 '-TGCTGGTGACAACCACGGCC-3' (SEQ ID NO: 8)), β-Actin (sense, 5 '-GTACTCCAGAAGACCAGAGG-3' (Denaturation at 55 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and annealing at 55 ° C for 30 seconds). The amplified product was amplified 28 times with extension, and the PCR product was obtained at 72 ° C (re-extension) and confirmed by 1% agarose gel electrophoresis.

(5) Osteoclast  culture

10% fetal bovine serum and penicillin-streptomycin were added to the α-MEM (modified eagle's medium) medium, and the cells were incubated at 37 ° C. in 5% And incubated in a CO ₂ incubator (MCO-18AIC, SANYO Co., Japan). (Sigma Chem. Co., St. Louis, Mo., USA) and 50 mg / ml vitamin C (Sigma Chem. Co., St. Louis, Mo.) in 10% FBS α-MEM medium for induction of differentiation. Louis, MO, USA) was used as a differentiation induction medium. The culture medium was changed every 3 days for the purpose of confirming the effect on proliferation, differentiation and mineralization for 27 days.

(6) Alkaline Phosphorylase by Osteoclast Culture Time alkaline  phosphatase activity

Saturated cultured MC3T3-E1 cells were adjusted to 1 × 10 ⁵ cells / well and dispensed into 96-well plates. After 24 hours, the cells were cultured for 24 hours in a differentiation-inducing medium. ALP enzyme activity was measured on days 3, 9, 18, and 27 while the polysaccharide derived from Oman and the positive control were added for every 27 days. After washing three times with PBS, 20 쨉 l of 0.1% Triton X-100 was added and the mixture was dissolved at 37 째 C for 30 minutes. 5 μl of the supernatant of the dissolved cells was used for quantification of the protein. 20 μl of 0.1 N glycine and 10 μl of 100 mM p-NPP (p-nitrophenylphosphate) were added to the remaining supernatant, followed by reaction at 37 ° C for 30 minutes. After the reaction, the reaction was stopped with 200 μl of 0.1N NaOH and absorbance was measured at 405 nm. The activity of ALP was determined by measuring the p-NP (p-nitrophenol) produced from p-NPP and plotting the activity of p-NP. The enzyme activity per unit protein content was calculated by dividing the activity by the amount of protein.

(7) alizarin - Red ( Alizarin - Red ) Calcification test by staining

The cultured MC3T3-E1 cells were adjusted to 1 × 10 ⁵ cells / mL and cultured on a plate. Differentiation induction medium and extracts were added for induction of calcification for 7 days. After incubation, the cells were fixed with 70% ethanol at 4 ° C for 1 hour. The concentration of the AR (Alizarin-Red) solution was adjusted to 40 mM in 10 mL of distilled water and then adjusted to pH 4.2. After fixation, cells were stained with AR solution (2 mL / well) for 10 minutes, washed 2 ~ 5 times with distilled water, and the non-stained portion was washed with PBS. The microscope was observed while wetting with PBS to prevent the surface from drying.

(8) Preparation of Gel for Prevention and Improvement of Bone Disease

The gel preparation for prevention and improvement of bone related diseases was composed of 64.3% of delonized water, 28% of ethanol, 3% of chamomile extract, 1% of purified polysaccharide derived from Oman, 1% of methanol, 0.5% of camphor, 0.5% of powder, 0.5% of corn oil extract, 0.5% of Carbopol 940 and 0.2% of triethanolamine, 0.2% of methylparaben and 0.2% of propylparaben as a preservative were mixed and sufficiently dispersed by stirring with a high pressure homogenizer of 1000 rpm or more. At this time, 0.1% of Blue No. 1 was added and then stirred slowly until the gelation was completed to prepare a gel for prevention and improvement of high-viscous bone disease (FIG. 2).

(9) Manufacture of ointment for prevention and improvement of bone diseases

For the prevention and improvement of bone related diseases, 72% of vaseline, 2% of shea butter and 3% of camphor were added to a beaker and dissolved at 80 ℃. 3% of methanol and 3% of squalane were added and then 2% of macadamia oil, 2% of avocado oil, 2% of jojoba oil, 2% of grape seed oil, 3% of evening primrose oil and 3% of coconut oil And the mixture was poured into a heat-resistant container made of polypropylene (PP) and placed at room temperature for about 30 minutes (FIG. 2).

Manufacture of gel and ointment for prevention and improvement of bone diseases Gel material Formulation ratio (%) Balm material Formulation ratio (%) Ultrapure water 64.3 Vaseline 72 ethanol 28 Shea Butter 2 Chamomile extract 3 camphor 3 Oman saponin refined water One Oman saponin refined water 3 Methanol One Methanol 3 camphor 0.5 Squalane 3 Aloe vera powder 0.5 Macadamia oil 2 Wild radish extract 0.5 Avocado oil 2 Carbopol 940 0.5 Jojoba oil 2 Triethanolamine 0.2 Grape seed oil 2 Methyl paraben 0.2 Evening Primrose Oil 3 Propylparaben 0.2 Coconut oil 3 Blue No. 1 0.1 Sum 100 Sum 100

(10) Measurement of stability of centrifugation

In order to confirm the stability by centrifugation in the stability analysis, 1 g of each sample was weighed and measured by Eppendorf tube, and centrifuged at 5,000 rpm and 10,000 rpm for 10 minutes through a centrifuge (1236MG, Gyrozen, Korea) . Changes in the gel and balm after the separation were visually observed.

(11) pH  Stability measurement

The pH was measured using a pH meter (Mettler Toledo, Switzerland) in which 2 g of gel and ointment were removed. The pH meter was connected to a power source for more than 10 minutes. It was immersed in distilled water beforehand. The detector was cleaned with distilled water and wiped off lightly.

(12) Storage stability test (light exposure test)

Gel and ointment were filled in transparent vinyl, respectively, and stored in a sunny place. Visual observation of changes such as discoloration, coloring, breaking, and rancidity for 15, 30, 60, 90 and 120 days Respectively.

(13) Stability test of product according to temperature

The stability test according to the temperature of this product is carried out by keeping the product at the specified temperature condition (refrigerated, normal temperature, 40 ℃), observing changes such as separation, precipitation and viscosity visually, The changes were observed at 15, 30, 60, 90, and 120 days, respectively.

Example  1: Nitric oxide ( Nitric oxide ) Production amount

NO is an important signaling molecule that protects against foreign substances in the immune system. It is produced by the action of NOS (nitric oxide synthase), which converts L-arginine into L-situululin and affects most tissue cells. It is known as a blood vessel relaxant, a neurotransmitter in the central nervous system, and a defense in the immune system.

In the present invention, RAW 264.7 cells were treated with polysaccharide and polysaccharide purified product (FIG. 3), and the concentration of polysaccharide purified SPF-Ⅰ, SPF-Ⅱ and SPF-Ⅲ from 0.1 ㎍ / mL to 6.94 to 9.80 μM . The amount of NO produced was significantly increased at 0.5 ㎍ / mL of SPF-Ⅰ, which was higher than that of other sections. The yield of LPS was 57.08%. NO is produced from cartilage cells of osteoblasts and joints. These results suggest that NO produced from osteoblasts and chondrocytes can regulate the activity of adjacent cells.

Example  2: cytokine ( Cytokine ) Production amount

(TNF-a) was measured in the culture supernatant of macrophages by treatment with polysaccharide purified water at a concentration of 0.05 to 0.5 占 퐂 / mL (FIG. 4). The results of the SPF-Ⅰ and SPF-Ⅰ sections showed higher than positive control (114.49% positive control) at 0.5 ㎍ / mL and 75.28% and 53.16% Respectively.

It is known that TNF-α is involved in the defense of the host against pathogens that enter the human body, and is also known to regulate the immune response of humans by inducing the secretion of cytokines such as IL-1, IL-6 and IFNs. , The ability of the omni polysaccharide and polysaccharide fractions to inhibit microbial and virus-sensitized cells significantly increased the activity of macrophages and various growth factors secreted from bone tissue Induced osteoclasts and osteoclasts by regulating the balance of activity. In particular, cytokine stimulating bone resorption, such as TNF-α, may promote osteoclast differentiation and activation from osteoblasts, mononuclear cells, and T-cells.

Example  3: Immune-related gene expression

After culturing Oman polysaccharide purified with 0.05 ~ 0.5 ㎍ / mL concentration of macrophage cell line, total RNA was extracted and reverse transcriptase cDNA was synthesized. The amount of mRNA expression was amplified by polymerase chain reaction (PCR) using primer The results are shown in Fig. As a result, expression of iNOS and COX-2 was not observed in the control group and expression was observed in LPS. Expression amount was increased with increasing concentration in SPF-Ⅰ, which is a polysaccharide purified product, . In addition, the amount of TNF-α and IL-6 mRNA expression for confirming cytokine production was confirmed to be the same as that of iNOS and COX-2. The purified polysaccharide from omnisporium promotes the production of NO, which may affect the activation of adjacent cells of osteoblasts and chondrocytes and the regulation of osteoblast and osteoclast activation, thereby regulating bone metabolism and calcification .

Example  4: Measurement of proliferation rate of osteoblast

Osteoblasts derived from undifferentiated mesenchymal cells are involved in the synthesis and calcification of bone matrix components. MC3T3-E1 cells, which are osteoblasts derived from mouse calvaria, are known to be differentiated into mesenchymal stem cells (mesenchymal stem cells) or connective tissue mesenchymalstem cells among various bone cell-derived cell lines. have. In addition, it belongs to the process of differentiation from osteoprogenitor to preosteoblast, osteoblast, and bonelining cell or osteocyte. It is a cell belonging to the process of proliferation, differentiation, calcification And is useful for studies related to the cell activity of bone cells. The osteoblast cell proliferation was evaluated by treating the polysaccharide-derived polysaccharide derived from Oman to a concentration of 0.025 ~ 1 / / mL, as shown in FIG. Cells were significantly more proliferated than the control group except for the 1 ㎍ / mL section of the polysaccharide purified product, and no significant difference was observed between polysaccharide purified products. The cell proliferation rate was significantly higher than that of the control group.

Example  5: Omani ALP ( alkaline 포스화제 ) Effect on activity

Alkaline phosphatase (ALP) is present in almost all tissues. In particular, ALP, which exists in the bone tissue, increases its activity when bone growth is active. Therefore, we investigated the effect of purified omal polysaccharide on ALP activity by measuring ALP activity in MC3T3-E1 osteoblasts as a biomarker for osteoblast activity. The osteoblast cells were cultured for 3 days, 9 days, 18 days, and 27 days, and the ALP activity was examined as a ratio to the control group. The result is shown in FIG. In the treatment of polysaccharide purified water, the ALP activity was higher than that of the control group on the third day of culture, and the ALP activity reached the highest level on the 18th day of culture and decreased thereafter. On the 18th day of SPF-Ⅰ, ALP activity was 177.15%, indicating that the activity was greatly increased, thereby promoting osteoblastic activity.

Example  6: Omani  Calcification Formability

Bone calcification is an important marker for osteoblast differentiation. Alizarin is a vegetable dye and has high adsorption power specifically for calcium. It is stained in the extracellular matrix of the mineralized cells, so the amount of mineralization is proportional to the degree of staining. The mineralization forming ability is shown in Fig. As a result, it was confirmed that the degree of staining and the formation of calcification were increased by osteoblast differentiation when SPF-Ⅰ, SPF-Ⅱ and SPF-Ⅲ were treated as compared with Oman saponin extract (SP). Therefore, it was confirmed that Oman saponin refined product induces proliferation of MC3T3-E1 osteoblast, increase of ALP activity, and formation of osteoblast differentiation and calcification.

Example  7: Omani  Derived polysaccharide Purified water  Evaluation of gel and night stability for preventing and improving joint disease

Gels and ointments for prevention and improvement of joint diseases were prepared and their stability was evaluated. The gels and ointments were prepared containing osmotic polysaccharide purified from the omnidia showing immunoenhancement effect and osteocyte proliferation effect.

(1) Measurement of stability of centrifugation

The results of analysis of stability according to the conditions of the product through the centrifugal separation test are shown in Table 2 below. After centrifugation at 5,000 rpm and 10,000 rpm for 10 minutes, both gel and balm products were not separated and showed a relatively stable appearance. It was observed that the oil phase and the functional material and the water layer were not separated and maintained a stable shape.

Result of centrifugation sample Gel type Ointment type 5,000 × 10 minutes ○ ○ 10,000 × 10 minutes ○ ○

○: stable, ●: unstable

(2) pH  And temperature stability measurement

Generally, the surface of human skin is adjusted to weak acidity and neutrality at pH 4.5 ~ 6.5. However, it is preferable to use neutral or weakly acidic cosmetics for skin because alkaline resistance weakens and bacterial growth causes skin diseases. The pH change should not be large and stable. The gel and ointment made from polysaccharide purified from Oman were measured at 4 ℃, RT (Room temperature), at 40 ℃ for 15, 30, 60, 90 and 120 days. The stability of the product was examined and the results are shown in Fig.

Gel and ointment products are contained in the range of pH 4.5 to 6.5 at 4 ° C, room temperature and 40 ° C, and are products for prevention and improvement of cartilage and joint diseases. there was. It can be confirmed that the pH change curves are similar to each other at each temperature condition, and it is found that the stability of the product is excellent.

(3) Storage stability test (light exposure test)

Gel and ointment were exposed to sunlight and fluorescent light directly or indirectly after the release and discolored. In order to prevent the change of quality characteristics of physical and chemical, The experimental results are shown in Table 3. In order to observe the change of gel and balm caused by light, the state of the state was observed visually for 120 days by keeping it in a sunny place. In the case of a gel product, a blue No. 1 was added The color of the product was blue when it was manufactured. When the product was added with vaseline as a main component, the color of the product was pale yellow. After 120 days of storage, the product remained stable without change in color and no change in odor and separation was observed. The color of all the products was not changed during the storage period of 120 days, and no change in separation, precipitation and viscosity was observed. It is believed that the stability of gel and ointment products for bone disease prevention and improvement prepared by containing polysaccharide purified from Oman is excellent.

Safety results of gel and chestnut products Storage days (days) 0 15 30 60 90 120 Gel type ○ ○ ○ ○ ○ ○ Ointment type ○ ○ ○ ○ ○ ○

○: stable, ●: unstable

<110> CATHOLIC UNIVERSITY OF DAEGU INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Composition for preventing or improving bone-related disease          containing polysaccharide purified from Styela plicata extract as          effective component <130> PN15027 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cccttccgaa gtttctggca gcagc 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggctgtcaga gcctcgtggc tttgg 25 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cactacatcc tgacccactt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 atgctcctgc ttgagtatgt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ttgacctcag cgctgagttg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cctgtagccc acgtcgtagc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gtactccaga agaccagagg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tgctggtgac aaccacggcc 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gtgggccgcc ctaggcacca g 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ggaggaagag gatgcggcag t 21

Claims (6)

Styela The present invention relates to a health functional food composition for preventing or ameliorating osteoarthritis related diseases, which comprises an extract of water and a protein hydrolyzing enzyme added to a plicata powder, and extracting the omal polysaccharide separated by ion exchange chromatography as an active ingredient. 2. The method according to claim 1, wherein the purified omal polysaccharide is a non-adsorbed fraction eluted with distilled water when eluted with a gradient of 0 to 1M salt concentration using ion exchange chromatography. &Lt; / RTI &gt; The health functional food composition according to claim 1, wherein the extract is extracted at 50 to 70 ° C for 12 to 18 hours. The method of claim 1, wherein the bone related disease is selected from the group consisting of osteoporosis, osteoarthritis, degenerative arthritis, knee arthritis, arthritis, systemic lupus erythematosus (SLE), spondyloarthritis, rheumatoid multiple muscle pain, ankylosing spondylitis, Inflammatory bone resorption diseases, calcium hydroxyapatite crystal precipitation diseases, and lime diseases. The present invention also relates to the use of the compounds of the present invention for the manufacture of a medicament for the treatment or prevention of osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, acute rheumatic fever, gout, cartilage calcification, periodontal disease caused by alveolar bone destruction, Wherein said composition is selected from the group consisting of: &lt; RTI ID = 0.0 &gt; (I) &lt; / RTI &gt; Styela The present invention relates to a pharmaceutical composition for the prevention or treatment of bone related diseases, which comprises extracting water and a protein hydrolyzing enzyme from plicata powder and extracting the purified omal polysaccharide separated by ion exchange chromatography as an active ingredient. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition is of gel or balm type.

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