Scapharca subcrenata extract and application thereof in preparation of product for preventing and / or treating osteoporosis

Abstract

The invention provides a scapharca subcrenata extract and application thereof in preparation of a product for preventing and / or treating osteoporosis, and belongs to the technical field of natural product extraction and application. Wherein the mass ratio of scapharca subcrenata protein peptide to scapharca subcrenata oligosaccharide in the scapharca subcrenata extract is (30-60): (0.01-10). Experiments prove that the prepared scapharca subcrenata extract can be beneficial to osteoblast proliferation and differentiation, can improve rat bone microstructure, promote bone formation, increase rat femoral bone dry weight and bone calcium content and effectively improve bone mineral density reduction, and has application prospects in preparation of products for preventing and treating osteoporosis.

Description

Technical Field

[0001] The invention relates to the technical field of natural product extraction and application, and in particular to a razor clam extract and application of the extract in preparing a product for preventing and / or treating osteoporosis. Background Art

[0002] Osteoporosis (OP) is a common bone disease in clinical practice and the bone metabolic disease with the highest incidence rate. The current prevalence rate in people over 50 years old in my country is 19.2%. There are many causes of osteoporosis, and the main clinical manifestations are bone loss, decreased bone density, decreased bone quality, bone microstructure destruction, increased bone brittleness, etc. Among them, bone density measurement is one of the indicators for evaluating osteoporosis, and a decrease in its value is also the most direct cause of osteoporosis. Scientific studies have shown that supplementing with appropriate substances can help slow down the rate of bone density reduction, which is conducive to the early prevention of osteoporosis and reducing the incidence of osteoporosis.

[0003] Arca subcrenata, commonly known as corrugated shellfish and hairy clam, is widely distributed in the western Pacific along the coasts of Japan, North Korea and China. It is also distributed in the coastal waters of my country, with the largest production in the coastal areas of Liaoning, Shandong and Hebei provinces. It is an important marine economic shellfish in my country. Arca subcrenata is not only delicious and tender, but also has high nutritional value. It contains protein, fat, carbohydrates, calcium, iron, phosphorus, iodine, vitamins, amino acids and taurine. Compared with other bivalve shellfish, Arca subcrenata has the nutritional advantages of high protein, low fat and low carbohydrates. According to a large number of ancient books and materials in my country, Arca subcrenata is not only a delicious food, but also a traditional Chinese medicine with a long history of medicinal use. Both the shell and the meat can be used as medicine. It is recorded in herbal books of various dynasties such as "Shennong's Herbal Classic" and "Suixiju Diet" that it has the effects of promoting blood circulation and removing blood stasis, reducing acid and relieving pain, replenishing blood, moistening the intestines, promoting body fluid and strengthening the stomach.

[0004] In recent years, studies on the biological activities of razor clams protein and razor clams sugar have appeared at home and abroad, but there are no reports proving the application of razor clams in improving bone density reduction and preventing and treating osteoporosis. Summary of the invention

[0005] In order to solve the above problems, this application proposes the following technical solutions:

[0006] On the one hand, the present application provides the use of a razor clam extract in the preparation of a product for preventing and / or treating osteoporosis, wherein the mass ratio of razor clam protein peptide to razor clam oligosaccharide in the razor clam extract is (30-60): (0.01-10).

[0007] In one embodiment, the mass ratio of razor clam protein peptide to razor clam oligosaccharide in the razor clam extract is (35-55): (1-5).

[0008] In one embodiment, the molecular weight of the razor clam oligosaccharide is 500-3000, preferably 650-1000.

[0009] In one embodiment, the molecular weight of the cristatin peptide is 200-5000 Da, preferably 400-800 Da.

[0010] In one embodiment, the razor clam extract is a powdered extract obtained by heating the razor clam with water and adding alkaline protease and α-amylase.

[0011] In one embodiment, the powdered extract is composed of scaly clam protein peptide and scaly clam oligosaccharide in a mass ratio of (35-55):(1-5).

[0012] In one embodiment, the product is selected from medicine, food, functional food, health food or special medical food.

[0013] In one embodiment, the product is used to promote osteoblast proliferation and differentiation, improve rat bone microstructure, increase bone dry weight and bone calcium content and / or improve decreased bone density.

[0014] In one embodiment, the scaly clam extract is prepared into a compound preparation with the same active ingredient or other active ingredients.

[0015] In one embodiment, the same active ingredient is selected from glucosamine and chondroitin sulfate.

[0016] In one embodiment, the other active ingredient is selected from hyaluronic acid.

[0017] In one embodiment, the dosage form of the product includes tablets, capsules, oral liquids, suspensions, granules, powders, pills, suppositories, injections, aerosols or drops.

[0018] In one embodiment, the administration route of the product includes oral administration, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, sublingual administration, nasal administration or aerosol administration.

[0019] On the other hand, the present application provides a method for preparing a scaly clam extract for preventing and / or treating osteoporosis, the method comprising:

[0020] Step 1: After mixing the razor clams and the extraction solvent, alkaline protease and α-amylase are added, and heating is performed for extraction;

[0021] Step 2: After the extraction is completed, the enzyme is inactivated, the supernatant is filtered, and the supernatant is dried to obtain the scaly clam extract.

[0022] In one embodiment, the razor clam powder is sieved through a 20-40 mesh sieve.

[0023] In one embodiment, the extraction solvent is water.

[0024] Optionally, the water may be purified water, deionized water or distilled water.

[0025] In one embodiment, the mass ratio of the razor clams to the extraction solvent is 1:(5-10), preferably 1:6.

[0026] In one embodiment, the addition amount of the alkaline protease is 0.1% to 10%, preferably 0.5% to 2%, and more preferably 1.5%.

[0027] The enzymatic activity of alkaline protease is 10,000 to 30,000 U / g, preferably 20,000 U / g.

[0028] In one embodiment, the added amount of the α-amylase is 0.1% to 10%, preferably 0.5% to 2%, and more preferably 1%.

[0029] The enzymatic activity of α-amylase is 5000 to 15000 U / g, preferably 10000 U / g.

[0030] It is to be understood that the percentage of enzyme addition is based on the mass of the raw material of razor clams.

[0031] In one embodiment, the heating extraction is performed at 40-60°C for 4-8 hours, preferably at 50°C for 6 hours.

[0032] In one embodiment, the enzyme inactivation can be performed by using a transient high temperature method to inactivate the added enzyme.

[0033] In one embodiment, the filtration may be performed by plate and frame filtration, with diatomaceous earth as the filtration aid.

[0034] In one embodiment, the drying may be performed by vacuum freeze drying or spray drying.

[0035] On the other hand, the present application also provides a razor clam extract prepared by the above method.

[0036] On the other hand, the present application also provides a product for preventing and / or treating osteoporosis, which contains 0.01% to 99.99% of the razor clam extract, preferably 0.01% to 50%; wherein the % is the mass volume percentage (w / v) or the mass percentage (w / w), preferably the mass percentage (w / w).

[0037] In one embodiment, the product is an injection, an oral solution or a tablet.

[0038] This application has at least the following beneficial effects:

[0039] The razor clam extract prepared by the specific extraction method of the present application contains a large amount of small molecular organic substances, which is conducive to the body's digestion and absorption, and also contains a specific mass ratio of razor clam protein peptides and razor clam oligosaccharides. The experimental results show that the razor clam extract is conducive to the proliferation and differentiation of osteoblasts, can improve the bone microstructure of rats, promote bone formation, increase the dry weight of rat femurs and bone calcium content, effectively improve the reduction of bone density, and has application prospects for the preparation of osteoporosis drugs, health foods and other products. In addition, the preparation method provided by the present application can simultaneously extract razor clam protein peptides and razor clam oligosaccharides, which can shorten the production process, save costs, and is suitable for industrial production. DETAILED DESCRIPTION

[0040] In order to more clearly explain the overall concept of the application, the following is described in detail by way of embodiment. In the following description, a large amount of specific details are provided so as to provide a more thorough understanding of the application. However, it is obvious to those skilled in the art that the application can be implemented without one or more of these details. In other examples, in order to avoid confusion with the application, some technical features well known in the art are not described.

[0041] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market. The test methods in the test experiments are all experimental methods known in the art.

[0042] The source information of the main materials, reagents and instruments involved in the following embodiments is as follows: hairy clam, purchased from Lianyungang Dingmai Food Co., Ltd.; alkaline protease, purchased from Beijing Donghua Qiangsheng Biotechnology Co., Ltd., with an enzyme activity of 20,000 U / g; α-amylase, purchased from Beijing Kewei Bo Biotechnology Co., Ltd., with an enzyme activity of 10,000 U / g; SD rats, purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.; plate and frame filter purchased from Shanghai Langdong Filtration Equipment Co., Ltd.; spray dryer, purchased from Shanghai Shunzhi Instrument Manufacturing Co., Ltd.

[0043] Example 1

[0044] This embodiment provides a razor clam extract, and the preparation method thereof is as follows:

[0045] 1. Raw material processing: grind the razor clam into powder, pass it through a 20-40 mesh sieve to obtain razor clam dry powder.

[0046] 2. Extraction: Use hairy clam powder as raw material, water as extraction solvent, and the solid-liquid ratio is 6:1. After soaking for 2 hours, heat and extract for a period of time. During the extraction, add alkaline protease and α-amylase to the extract and stir evenly.

[0047] 3. Enzyme inactivation: After the enzymatic hydrolysis is completed, the added enzyme is inactivated by instantaneous high temperature method, and the solution is quickly cooled after inactivation.

[0048] 4. Filtration: The solution after enzyme inactivation is filtered by plate and frame filtration method with diatomaceous earth as the filter aid to remove the residue and obtain the supernatant.

[0049] 5. Drying: The filtered solution is subjected to vacuum freeze drying or spray drying to obtain a mixture powder consisting of razor clam oligosaccharides and razor clam protein peptides.

[0050] The above method was used to prepare a series of sample examples 1# to 10# of razor clam extract. The difference between the different sample examples is that the temperature, time, enzyme amount and ratio used in the preparation are slightly different. The specific parameters are shown in Table 1.

[0051] The structures and molecular weights of the scaly clam oligosaccharides and scaly clam protein peptides in the prepared sample powder were tested and confirmed, and the molecular weights of the scaly clam oligosaccharides were measured to be 650-1000, and the molecular weight of the scaly clam protein peptides were measured to be 400-800Da.

[0052] At the same time, the contents of sclerotinia protein peptide and sclerotinia oligosaccharide in the series of samples were detected, wherein the content of sclerotinia protein peptide was detected by using BCA protein concentration determination kit; the content of sclerotinia oligosaccharide was detected by high performance liquid chromatography HPLC, and the chromatographic conditions were as follows:

[0053] Chromatograph: LC-20AT high performance liquid chromatograph (Shimadzu);

[0054] Chromatographic column: FUJI NH-SG (4.6 mm × 250 mm, 5 μm);

[0055] Mobile phase: acetonitrile / water (65 / 35);

[0056] Flow rate: 1.0 mL / min;

[0057] Column temperature: 30°C;

[0058] Detector: RID-20AT differential detector.

[0059] Example 2

[0060] In this example, a series of razor clam extracts prepared in Example 1 were used to verify the efficacy of improving bone density reduction. The specific method is as follows:

[0061] Female adult rats were used. After the ovaries were removed, bone metabolism was enhanced and changes occurred in which bone reabsorption (osteoclasts) was greater than bone formation (osteogenesis). This change manifests as bone loss, which can lead to a model of reduced bone density after a certain period of accumulation. While establishing the model, the model group rats were supplemented with samples of razor clam extract. Through the bone metabolism regulating effects of razor clam extract such as inhibiting osteoclasts or promoting osteogenesis, the effect of increasing bone density and bone calcium content was observed, thereby evaluating the function of razor clam extract samples in improving bone density.

[0062] Three-month-old adult female SD rats were used. After adaptive feeding, 10 rats underwent sham surgery (similar surgery but without bilateral ovarian removal, only partial fat removal), and the remaining rats underwent bilateral ovariectomy to establish the model. Three days after the modeling surgery, the rats were randomly divided into a scaly clam extract group and a positive drug control group according to their body weight, with 10 rats in each group. The test substance or distilled water was given by gavage at a dose of 400 mg / kg / d, once a day, for 3 months, during which the animals were free to drink water and eat. After the end of the administration, the right femur was taken to measure the bone dry weight and calcium content in the bone sample, and the left femur was taken for bone density measurement.

[0063] The preparation parameters, protein peptide and oligosaccharide contents, and bone calcium and bone density of Examples 1# to 10# and the remaining groups are shown in Table 1. It can be understood that the percentage of protein peptide and oligosaccharide in Table 1 is the percentage of the mass of the raw material of razor clams (or can be understood as the yield), that is, the mass ratio of protein peptide and oligosaccharide in the obtained razor clams extract powder.

[0064] Table 1

[0065]

[0066] Note: *Compared with the model group, P<0.05; #Compared with the sham operation group, P<0.05

[0067] The results in Table 1 show that compared with the sham operation group, all indicators of the group containing the razor clam extract were significantly improved, indicating that the prepared razor clam extract has the effect of improving the decreased bone density in the ovariectomized model rats.

[0068] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. Use of a razor clam extract in the preparation of a product for preventing and / or treating osteoporosis, characterized in that: The mass ratio of scaly clam protein peptide to scaly clam oligosaccharide in the scaly clam extract is (30-60): (0.01-10).

2. The use according to claim 1, characterized in that: The molecular weight of the scaly clam oligosaccharide is 500-3000 Da; and / or the molecular weight of the scaly clam protein peptide is 200-5000 Da.

3. The use according to claim 1, characterized in that: The product is selected from medicines, foods, functional foods or special medical foods.

4. The use according to claim 1, characterized in that: The product is used to promote osteoblast proliferation and differentiation, improve rat bone microstructure, increase bone dry weight and bone calcium content and / or improve decreased bone density.

5. The use according to claim 1, characterized in that: The razor clam extract is prepared into a compound preparation with the same active ingredient or other active ingredients, wherein the same active ingredient is selected from glucosamine and chondroitin sulfate; and the other active ingredient is selected from hyaluronic acid.

6. The use according to claim 1, characterized in that: The dosage forms of the product include tablets, capsules, oral liquids, suspensions, granules, powders, pills, suppositories, aerosols or drops.

7. A method for preparing a scaly clam extract for preventing and / or treating osteoporosis, characterized in that: The method comprises: Step 1: After mixing the razor clams and the extraction solvent, alkaline protease and α-amylase are added, and heating is performed for extraction; Step 2: After the extraction is completed, the enzyme is inactivated, the supernatant is filtered, and the supernatant is dried to obtain the scaly clam extract.

8. The method according to claim 7, characterized in that The razor clam powder is sieved through a 20-40 mesh sieve; And / or, the extraction solvent is water; And / or, the mass ratio of the razor clams to the extraction solvent is 1:(5-10); And / or, the enzymatic activity of the alkaline protease is 10000-30000 U / g, and the added amount is 0.1%-10%, and the enzymatic activity of the α-amylase is 5000-15000 U / g, and the added amount is 0.1%-10%; And / or, the heating extraction is extraction at 40-60° C. for 4-8 hours.

9. The razor clam extract prepared by the method according to claim 7 or 8.

10. A product for preventing and / or treating osteoporosis, characterized in that: The product contains 0.01% to 99.99% of the razor clam extract as claimed in claim 9.

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