TWI617315B - Bass extract with an effect on the stimulation and maintenance of bone - Google Patents

Abstract

The invention provides a sea bass extract, which reacts with an enzyme through an extraction process to produce a peptide having a specific molecular weight in a specific segment. The sea bass extract of the present invention has the effects of promoting the health of bone cells and the proliferation of collagen, and can be used for preparing bone health products.

Description

Bone-protecting sea bass extract

The invention provides a sea bass extract having a bone-protecting effect, particularly a sea bass extract peptide having a bone-protecting effect.

Human bone density depends on the balance between the growth and differentiation of osteoclast and osteoblast cells in the bone. Osteoclasts cause bone erosion, while osteoblasts promote bone formation. Postmenopausal women and the elderly are the main cause of bone loss due to the increased number of osteoclasts and excessive activation, which accelerates bone erosion. Therefore, osteoporosis has been a difficult symptom for middle-aged and elderly people at home and abroad, especially for women after menopause. Joint pain and fractures caused by osteoporosis not only plague most middle-aged and elderly people, but also a heavy burden on medical resources. Up to now, most of the health foods in bone maintenance have only been used to passively supplement calcium or increase calcium absorption, that is, the more advanced glucosamine, which also only reduces joint pain. In the clinic, there are medicines for treating osteoporosis, but these medicines have some other side effects.

Furthermore, bone defect repair has been an important subject in clinical medical research for a long time. So far, there has been no effective solution to the problem of repairing large-scale bone defects caused by trauma, infection, and tumor resection. Because the source of autogenous bone is limited and it will cause great pain to patients, other bone repair materials, such as allogeneic bone or xenogeneic bone, are likely to cause problems of immunity and disease transmission.

Sea bass is rich in nutrients such as unsaturated fatty acids, proteins, peptides, amino acids and vitamins, and according to animal experiments, it can promote the ability of rat skin wounds to heal. In addition, Taiwanese seabass produced 26,094 tons in 2013, with a total output value of 2.18 billion yuan. If this seabass is used, it can increase the added value of Taiwan's fisheries.

In view of this, the present invention extracts components with bone-protecting effects from sea bass, which have the effects of promoting wound healing, collagen proliferation and bone-protecting, and at the same time non-toxic and no side effects, good quality, high safety, low cost, The advantages of easy absorption can better meet the needs of osteoporosis patients or postoperative repairers.

The invention provides a composition for a bone-care health product, which comprises a sea bass extract, wherein the sea bass extract has a peptide having a molecular weight between 1000 Da and 8000 Da.

In one embodiment of the present invention, the sea bass extract contains at least 30% of peptides.

In an embodiment of the present invention, the peptide distribution of the sea bass extract is 8% to 12% of the peptide having a molecular weight between 5000Da and 6000Da, and 8% to 12% of the peptide having a molecular weight between 6000Da and 7000Da. And 12% to 16% of peptides having a molecular weight between 7000Da and 8000Da; further comprising 5% to 8% of peptides having a molecular weight between 1000Da and 2000Da, and 15% to 18% having a molecular weight between 2000Da and 3000Da Peptides with a molecular weight of 7% to 10%, peptides with a molecular weight between 3000Da and 4000Da, and peptides with a molecular weight between 12% and 15% between 4000Da and 5000Da.

In an embodiment of the present invention, the sea bass extract is obtained by extracting fish skin, scales, fish bones and fish meat of sea bass.

In an embodiment of the present invention, the sea bass extract is selected from the group consisting of Keratinase, Trypsin, Bromelain, and Papain. (Papain), Pepsin, Alcalase, Neutrase, Protamex, and Flavourzyme.

In one embodiment of the present invention, the sea bass extract has the effect of promoting collagen proliferation and bone cell proliferation.

In one embodiment of the present invention, the composition for a bone-care health care product is a tablet, capsule, liquid, gel, slurry, suspension, powder pack, dressing, emulsion, spray, film, In the form of a salve or patch.

In one embodiment of the present invention, the composition for a bone-protection health product is a beverage, food, or external article.

The present invention also provides a use of the sea bass extract of the present invention for preparing a bone-protecting health product.

In order to effectively increase the added value of sea bass, the sea bass extract with a special molecular weight is produced through the reaction of the extraction process with enzymes. The sea bass extract has good effects on bone cell health and collagen proliferation. It is suitable for development as a health care material in the future. Consumption for frail or post-operative repairs can promote wound healing, collagen proliferation and bone protection.

The first figure is a data chart of comparative analysis of molecular weight distributions of the sea bass extract of the present invention, the commercially available sea bass essence and the commercial sea bass soup.

The second figure is a data chart of comparative analysis of the peptide content of the sea bass extract of the present invention, the commercially available sea bass essence and the commercial sea bass soup.

The third graph is a data chart of the percentage of cartilage differentiation in the bass extract of the present invention and the control group.

The fourth graph is a data chart of the percentage of collagen proliferation of the sea bass extract and the control group of the present invention.

The present invention uses sea bass as a material, and reacts with an enzyme to produce peptides with a specific molecular weight through an extraction process. Cell experiments have confirmed that the sea bass extract of the present invention can promote bone cell proliferation up to 5.2 times and can promote collagen proliferation. Its proliferation was 4.4 times that of the control group. Therefore, the sea bass extract of the present invention can promote wound healing, collagen proliferation and bone protection.

Example 1 Preparation method of sea bass extract of the present invention

The process of extracting sea bass with bone-protecting effect according to the present invention is as follows: 1. The sea bass is cut to a size of about 2 to 6 cm by a slicer as a raw material, wherein the sea bass comprises fish skin, scales, fish bones and fish meat; 2. 2 to 5 Double the volume of RO reverse osmosis water to clean the raw materials, and repeatedly clean the seabass 2 to 3 times to remove blood water and impurities; 3. Perform acid treatment at a ratio of 1: 5 seabass to acid solution (w / v), of which the type of acid Soak with hydrochloric acid, sulfuric acid or phosphoric acid at a concentration of 1 to 5% for 10 to 48 hours at 15 to 20 ° C. 4. Wash the raw materials after acid treatment with 2 to 5 times the volume of RO reverse osmosis water, and repeat cleaning 2 to 3 Deacidification is performed twice; 5. Adjust the pH value between 4 and 8 with calcium carbonate; 6. Extraction with hot water at 55 to 100 ° C for 1 to 6 hours; 7. Centrifuge at 3000r.pm for 10 minutes and perform deslagging filtration ; 8. Use 0.2-1% calcium carbonate or lime to remove impurities and clarify; 9. Filter with 1-10 μm filter membrane; 10. Adsorption filtration with ion exchange resin column to remove impurities and lime, and further isolate and purify the effective peptide; 11. Deodorize and decolorize by activated carbon; 12. Concentrate under reduced pressure at 50 to 60 ° C, and concentrate 10 To 20 times; 13. adding a complex enzyme for hydrolysis at 40 to 60 ° C for 1 to 5 hours, wherein the complex enzyme contains 0.01% -0.1% keratinase and bromelain; 0.1- 5% Papain, Trypsin and Pepsin, 0.1-5% Alcalase, Neutrase, Protamex, and Flavourzyme 14. Inactivation of the enzyme at 85 to 95 ° C for 10 to 30 minutes; inactivation of the enzyme; 15. cooling; 16. filtration with diatomaceous earth and activated carbon to clarify and deodorize the peptide extract 17. filtration; 18. Ultra-high temperature (UHT) sterilization at 135 to 140 degrees C for 3 to 5 seconds; 19. 0.2 μm filter membrane to filter bacteria and remove small impurities to obtain the bass extract of the present invention.

Example 2 Detection of the molecular weight of the sea bass extract of the present invention

The present invention compares and analyzes the sea bass extract obtained in Example 1 with commercially available sea bass essence and commercial sea bass soup, and uses Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry MALDI -TOF) (model Voyager-DE PRO, brand Applied Biosystems) The peptide molecular weight distribution was analyzed. As shown in the first figure, the molecular weight of the sea bass extract of the present invention is less than 8,000 Da, and the commercially available sea bass essence and the sea bass soup contain large-molecular-weight proteins, which are less easily absorbed by the human body.

Furthermore, the molecular weight distribution of the sea bass extract of the present invention is further analyzed, which has a peptide having a molecular weight between 1000 Da and 8000 Da, of which 5% to 8% is a peptide having a molecular weight between 1000 Da and 2000 Da, and 15% to 18% peptides between 2000 Da and 3000 Da molecular weight, 7% to 10% peptides between 3000 Da and 4000 Da molecular weight, 12% to 15% peptides between 4000 Da and 5000 Da molecular weight, 8% To 12% of peptides having a molecular weight between 5000Da to 6000Da, 8% to 12% of peptides having a molecular weight between 6000Da to 7000Da, and 12% to 16% of peptides having a molecular weight between 7000Da to 8000Da (No. A picture).

In addition, in the analysis of peptide content, the sea bass extract of the present invention has a peptide content of 32%, and the total peptide content of the sea bass extract is 28 to 100 times more than that of commercially available sea bass soup and commercially available sea bass essence. As shown in the second figure, the sea bass extract after the extraction and purification of the present invention can obtain the best total peptide content, which is more conducive to the repair and healing of cartilage and hard bone damage.

Example 3 Test for promoting chondrocyte ( in vitro ) differentiation of sea bass extract of the present invention

In order to confirm that the sea bass extract of the present invention has the ability to promote the growth of chondrocytes, the present invention uses Alcian blue staining (Sigma-Aldrich, St. Louis, MO) to confirm the sea bass extract obtained in Example 1. Ability of chondrogenic differentiation.

The invention uses the sea bass extract to treat ATDC5 chondrocyte cell line (obtained from the Japanese RIKEN public database), and uses untreated human chondrocyte cell line as a control group to observe that the sea bass extract secretes cartilage matrix to human chondrocyte cell line ( GAG). The results show that, Analysis by Elson blue staining showed that human chondrocytes treated with the sea bass extract could enhance cartilage matrix (GAG) secretion. The sea bass extract also increased cell survival rate, thereby reducing the chance of cartilage tissue damage. Moreover, the analyzed data results are shown in the third figure. Compared with the percentage of cartilage differentiation of the control group not treated with sea bass extract, the human chondrocyte cell line treated with the sea bass extract of the present invention was treated by Elson. The percentage of cartilage differentiation after blue staining is 521%. Therefore, the human chondrocyte cell line treated with the bass extract of the present invention can effectively increase the cartilage differentiation ability by 5.21 times, confirming that the bass extract of the present invention has the ability to effectively promote the growth of chondrocytes.

Example 4 Test of Proliferation of Collagen by CCD-966SK Human Fibroblasts by the Sea Bass Extract of the Present Invention

In order to confirm that the sea bass extract of the present invention has the ability to promote the proliferation of cellular collagen, the present invention uses the sea bass extract to treat CCD-966SK human fibroblasts (purchased from a food factory). After processing, the extract is analyzed for collagen The ability of protein to proliferate. The collagen content was analyzed by Sircol ^TM Collagen Assay S1111, which can be used for affinity binding to the triple helix structure of collagen. The comparative analysis of human fibroblasts treated with sea bass extract and without sea bass extract was performed to observe the promotion of the sea bass extract. The ability of collagen to proliferate.

Using the Sircol ^™ method, human skin fibroblasts were seeded into a 24-well plate at 4 × 10 ⁴ / well, and after 24 hours of culture, the supernatant was collected to determine the collagen concentration. The measuring principle is to use the red dye Sirius red to connect with the [Gly-XY] _n structure of collagen. First, add Sirius red to the collected supernatant, and shake it at room temperature for 30 minutes to react the Sirius red and the supernatant. The collagen was bound, and centrifuged at 10,000 × g for 10 minutes. The unbound Sirius red was discarded. The centrifuged precipitate was dissolved in an alkaline solution, and the concentration of collagen was analyzed at an absorbance of 540 nm. The results are shown in the fourth figure. Compared to the human collagen control group of human fibroblasts not treated with the sea bass extract, the collagen percentage is 100%, and the collagen of the human fibroblast control group treated with the sea bass extract of the present invention is 100%. The percentage can be increased to 439%. Therefore, the ability of the sea bass extract of the present invention to promote collagen proliferation is 4.39 times that of the control group, confirming that the sea bass extract of the present invention has a good effect in promoting collagen increase.

In summary, the sea bass extract of the present invention can promote osteoblast proliferation by 5.2 times and 4.4 times of collagen proliferation, which confirms that the sea bass extract has the efficiency of increasing chondrocyte survival, cartilage differentiation, and promoting collagen proliferation. The ability to achieve the effect of bone protection, so it can be used in the preparation of bone health products, in which the bone health products can be in the form of drinks or food, etc., in order to solve the busy life of modern people and difficult to cook sea bass problem.

Therefore, the sea bass extract of the present invention improves the utilization value of sea bass, and has the advantages of non-toxicity and no side effects, good quality, high safety, low cost, easy absorption and the like; meanwhile, the sea bass extract has ingredients for protecting bones to achieve The effect of promoting bone cell health and collagen proliferation can better meet the needs of osteoporosis or postoperative repairers.

Claims (6)

A perch extract is used for preparing a drug for promoting chondrocyte differentiation, wherein the perch extract is obtained by non-high pressure extraction of fish skin, scales, fish bones and fish meat of perch, and has a molecular weight of 1000Da to 8000Da Peptide. The use according to item 1 of the scope of patent application, wherein the sea bass extract contains at least 30% of peptides. The use according to item 1 of the patent application range, wherein the sea bass extract contains 8% to 12% peptides having a molecular weight between 5000Da and 6000Da, and 8% to 12% peptides having a molecular weight between 6000Da and 7000Da. Peptide and 12% to 16% peptides with molecular weights between 7000 Da and 8000 Da. The use according to item 3 of the patent application range, wherein the sea bass extract further contains 5% to 8% peptides having a molecular weight between 1000 Da and 2000 Da, and 15% to 18% having a molecular weight between 2000 Da and 3000 Da. Peptide, 7% to 10% peptide having a molecular weight between 3000Da and 4000Da, and 12% to 15% peptide having a molecular weight between 4000Da and 5000Da. The use according to item 1 of the patent application scope, wherein the sea bass extract is selected from the group consisting of Keratinase, Trypsin, Bromelain, Papain, and Pepsin. ), Alkaline protease (Alcalase), neutral protease (Neutrase), complex protease (Protamex) and flavor protease (Flavourzyme) group of extraction. The use as described in item 1 of the patent scope, wherein the drug is a tablet, capsule, liquid, gel, slurry, suspension, powder pack, dressing, emulsion, spray, film, ointment or patch Form.