Astaxanthin and fish oil compound for assisting in reducing blood sugar as well as preparation method and application of astaxanthin and fish oil compound

Abstract

The invention belongs to the technical field of medicines, and discloses an astaxanthin and fish oil compound for assisting in reducing blood sugar as well as a preparation method and application of the astaxanthin and fish oil compound. The astaxanthin and fish oil compound for assisting in reducing blood sugar comprises the following raw materials: astaxanthin oil and fish oil. The invention also discloses an application of the astaxanthin and fish oil compound for assisting in reducing blood sugar in preparing a medicine for reducing blood sugar. The astaxanthin and fish oil compound prepared by the invention is simple in component, high in antioxidant activity and remarkable in blood sugar reducing effect, and has a wide application prospect in the aspect of treating diabetes mellitus.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to an astaxanthin fish oil complex for assisting in lowering blood sugar, and a preparation method and application thereof. Background Art

[0002]

[0003] Astaxanthin (ASTA) is called 3,3'-dihydroxy-β-carotene-4,4'-one, and its chemical formula is C 40 H 52 O4 is an orange-red fat-soluble carotenoid with a molecular weight of 536.87. As a powerful natural antioxidant, astaxanthin has shown significant blood sugar lowering potential in many animal experiments. Astaxanthin is 1000 times more antioxidant than vitamin E and is considered to be a "super vitamin E". It is safe and non-toxic to the human body and has been widely used in functional foods, health products and medicines. At present, the product positioning of astaxanthin functional foods and medicines is mainly to strengthen the immune system function and prevent oxidative damage to blood LDL-cholesterol. However, there is still no blood sugar lowering astaxanthin product. A large number of experiments have shown that the antioxidant property of astaxanthin can inhibit the oxidative stress response during the development of diabetes, thereby inhibiting the increase of blood sugar and the occurrence of diabetic complications. Haematococcus pluvialis is the biological resource with the highest astaxanthin accumulation level in nature, and is also a new resource food approved by the Ministry of Health. Astaxanthin in Haematococcus pluvialis mainly exists in the form of monoester or diester mixture. Orally taken astaxanthin esters are enzymatically hydrolyzed into free astaxanthin in the small intestine, and their biological efficacy is the same as that of free astaxanthin.

[0004] Fish oil is a kind of oil extracted from fish. It is rich in polyunsaturated fatty acids, mainly containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Fish oil has become one of the most respected health products today because of its unique effects in preventing and treating cardiovascular diseases, regulating blood lipids, and anti-inflammation. Studies have shown that fish oil rich in DHA and EPA has significant therapeutic effects in controlling blood sugar levels and improving diabetic complications.

[0005] At present, there is no effective way to prevent and treat diabetes at home and abroad, and drug therapy is still the main treatment method. However, drug therapy cannot cure the disease, and it has certain toxic side effects, and makes patients dependent on drugs, which seriously affects the quality of life of diabetic patients. Studies have shown that oxidative stress is a common pathogenesis of various complications of diabetes. Antioxidants can inhibit the production of a large amount of reactive oxygen, improve the body's antioxidant defense system, and have a significant effect on diseases caused by oxidative stress. The use of antioxidants to prevent and treat diabetes has become a research hotspot. However, the various astaxanthin and fish oil products on the market currently have shortcomings such as a single active ingredient and unclear effects, which makes it difficult to achieve ideal results.

[0006] For example, Chinese patent CN104082751A discloses a deep-sea fish oil soft capsule containing astaxanthin, the main active ingredients of which are: deep-sea fish oil [containing EPA (eicosapentaenoic acid), DHA (docosahexaenoic acid)], astaxanthin oil (containing astaxanthin). The main auxiliary materials are water, vitamin E, glycerin and edible animal gelatin. The fish oil soft capsule effectively combines -3 highly unsaturated fatty acids (EPA, DHA) and astaxanthin with strong antioxidant effects, and the components further synergize to effectively prevent cardiovascular and cerebrovascular diseases, improve immunity, remove free radicals in the body, and play a role in delaying aging. However, the effect of the product in this application on lowering blood sugar needs to be studied, and its antioxidant properties are not clearly stated.

[0007] Another example is Chinese patent CN104984102A, which discloses an astaxanthin health capsule with hypoglycemic effect. It is scientifically compatible with astaxanthin and Chinese herbal medicine raw materials, preferably 30-40 parts of astaxanthin, 15-20 parts of bitter melon, 15-18 parts of wolfberry, 10-14 parts of corn silk, 8-10 parts of cooked rehmannia, 8-10 parts of liquorice, 6-10 parts of schisandra, 4-8 parts of atractylodes, 5-8 parts of Lycium bark, 4-6 parts of platycodon, 4-6 parts of anemarrhena, 4-6 parts of black plum, 3-5 parts of dodder seed, 2-3 parts of stevia, and then prepared by the process of deployment, mixing, sterilization, and filling capsules. The product has significant hypoglycemic, qi-tonifying, strengthening the body, anti-fatigue, and longevity effects through the synergistic effect between astaxanthin and Chinese herbal medicine. It is safe and has no side effects, is easy to eat, and has a good market application prospect. However, the product ingredients of this application are complex, and the antioxidant activity needs to be studied.

[0008] Therefore, it is particularly important to develop a product that combines astaxanthin and fish oil to achieve synergistic effects and is safe and effective, while also having auxiliary blood sugar lowering and antioxidant functions. Summary of the invention

[0009] Based on the problems existing in the prior art, the present invention provides an astaxanthin fish oil complex for assisting in reducing blood sugar, and its preparation method and application, and clarifies the antioxidant and hypoglycemic effects of the complex through HepG2 cell antioxidant experiment and hypoglycemic experiment. The key raw material used in the complex is astaxanthin oil, which comes from Haematococcus pluvialis astaxanthin oil (astaxanthin content 10%), and Haematococcus pluvialis is recognized as the natural raw material richest in astaxanthin in the world.

[0010] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0011] The present application provides an astaxanthin fish oil complex for assisting in lowering blood sugar, characterized in that it comprises the following raw materials: astaxanthin oil and fish oil, wherein the mass ratio of the astaxanthin oil to the fish oil is 0.5-2:10-20.

[0012] Preferably, the mass ratio of astaxanthin oil to fish oil is 1-1.5:12-18;

[0013] More preferably, the mass ratio of astaxanthin oil to fish oil is 1-1.2:12-15;

[0014] Further preferably, the mass ratio of astaxanthin oil to fish oil is 1:12.

[0015] The present application also provides a method for preparing an astaxanthin fish oil complex for assisting in lowering blood sugar, comprising the following steps: fully stirring the astaxanthin oil and fish oil in the above-mentioned specific ratio to make them uniformly mixed.

[0016] The present application also provides an application of an astaxanthin fish oil complex for assisting in lowering blood sugar in the preparation of a blood sugar lowering drug.

[0017] Preferably, the dosage form of the drug includes but is not limited to: capsules, granules, tablets, oral liquid or pills.

[0018] The present application also provides an astaxanthin fish oil capsule for assisting in lowering blood sugar, characterized in that the contents include an astaxanthin fish oil complex and vitamin E.

[0019] Preferably, the preparation method of the astaxanthin fish oil capsule for assisting in lowering blood sugar comprises the following steps:

[0020] (1) Preparation of contents: Mix astaxanthin oil and fish oil, add vitamin E, and mix well;

[0021] (2) Preparation of capsule skin material: Dissolve gelatin, glycerin and water and degas;

[0022] (3) The contents and capsule skin are compressed to prepare astaxanthin fish oil soft capsules.

[0023] Preferably, the mass ratio of astaxanthin oil, fish oil and vitamin E in step (1) is 0.5-2:10-20:0.1-1;

[0024] More preferably, the mass ratio of astaxanthin oil to fish oil in step (1) is 1-1.5:12-18:0.2-0.8;

[0025] Further preferably, the mass ratio of astaxanthin oil to fish oil in step (1) is 1-1.2:12-15:0.4-0.6;

[0026] More preferably, the mass ratio of astaxanthin oil to fish oil in step (1) is 1:12:0.5.

[0027] Preferably, the volume ratio of gelatin, glycerol and water in step (2) is 1:0.4:1.

[0028] Preferably, the dissolving in step (2) is performed by heating, and the heating temperature is 80°C.

[0029] Preferably, the degassing in step (2) is carried out by ultrasonic degassing.

[0030] Preferably, the compression in step (3) is performed by a soft capsule compression method.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention combines astaxanthin and fish oil, which has simple ingredients and certain synergistic effects;

[0033] (2) The preparation process of the present invention is simple and suitable for large-scale production;

[0034] (3) The present invention has both antioxidant and hypoglycemic effects and has broad application prospects in the treatment of diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The effect of astaxanthin fish oil complex on the activity of cell SOD;

[0036] Figure 2 The effect of astaxanthin fish oil complex on MDA content;

[0037] Figure 3 The effect of astaxanthin fish oil complex on protein carbonyl content;

[0038] Figure 4 The effect of astaxanthin fish oil complex on GSH content;

[0039] Figure 5 The effect of astaxanthin fish oil complex on glucose consumption;

[0040] Note: Group settings (1: blank group, 2: model group; 3: positive group; 4: Example 1-low concentration dose group; 5: Example 1-medium concentration dose group; 6: Example 1-high concentration dose group; 7: Example 2-medium concentration dose group; 8: Example 3-medium concentration dose group; 9: Example 4-medium concentration dose group; 10: Comparative Example 1-low concentration dose group; 11: Comparative Example 1-medium concentration dose group; 12: Comparative Example 1-high concentration dose group; 13: Comparative Example 2-low concentration dosage group; 14: comparative example 2-medium concentration dosage group; 15: comparative example 2-high concentration dosage group; 16: comparative example 3-medium concentration dosage group; 17: comparative example 14-medium concentration dosage group) In the figure, compared with the blank control group, ## represents P<0.01, # represents P<0.05; compared with the model group, **** represents P<0.0001, *** represents P<0.001; ** represents P<0.01; * represents P<0.05. DETAILED DESCRIPTION

[0041] The technical scheme of the present invention is clearly and completely described below with specific implementation methods. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The materials, reagents, etc. used, if not otherwise specified, are reagents and materials available from commercial sources.

[0042] Example 1: An astaxanthin fish oil complex for assisting in lowering blood sugar

[0043] Including the following ingredients:

[0044] Astaxanthin oil 1g, fish oil 12g

[0045] Example 2: An astaxanthin fish oil complex for assisting in lowering blood sugar

[0046] Including the following ingredients:

[0047] Astaxanthin oil 0.5g, fish oil 10g

[0048] Example 3: An astaxanthin fish oil complex for assisting in lowering blood sugar

[0049] Including the following ingredients:

[0050] Astaxanthin oil 2g, fish oil 20g

[0051] Example 4: An astaxanthin fish oil complex for assisting in lowering blood sugar

[0052] Including the following ingredients:

[0053] Astaxanthin oil 1g, fish oil 15g

[0054] Comparative Example 1: An astaxanthin fish oil complex for assisting in lowering blood sugar

[0055] Including the following ingredients:

[0056] Fish oil 12g

[0057] Comparative Example 2: An astaxanthin fish oil complex for assisting in lowering blood sugar

[0058] Including the following ingredients:

[0059] Astaxanthin oil 1g

[0060] Comparative Example 3: An astaxanthin fish oil complex for assisting in lowering blood sugar

[0061] Including the following ingredients:

[0062] Astaxanthin oil 0.2g, fish oil 0.8g

[0063] Comparative Example 4

[0064] According to the method described in the reference document CN104082751A, an astaxanthin fish oil complex was prepared. The specific preparation method is as follows:

[0065] The raw materials deep-sea fish oil and astaxanthin oil are mixed together in a certain ratio and stirred evenly. The net content of the effective ingredients in the contents is as follows: EPA: 30%, DHA: 20%, astaxanthin oil: 2%.

[0066] Effect experiment

[0067] 1. Antioxidant test

[0068] (1) Experimental methods:

[0069] First, the astaxanthin fish oil complexes prepared in Examples 1-4 and Comparative Examples 1-4 were ultrasonically mixed in 1 mL of DMSO, and then diluted with DMEM medium to the corresponding concentration to obtain Example 1-low concentration dosage group (astaxanthin concentration of 30 μg / mL), Example 1-medium concentration dosage group (astaxanthin concentration of 60 μg / mL), Example 1-high concentration dosage group (astaxanthin concentration of 120 μg / mL), Example 2-medium concentration dosage group, Example 3-medium concentration dosage group, Example 4-medium concentration dosage group, Comparative Example 1-low concentration dosage group, Comparative Example 1-medium concentration dosage group, Comparative Example 1-high concentration dosage group, Comparative Example 2-low concentration dosage group, Comparative Example 2-medium concentration dosage group, Comparative Example 2-high concentration dosage group, Comparative Example 3-medium concentration dosage group, Comparative Example 4-medium concentration dosage group.

[0070] Digest HepG2 cells to prepare cell suspension and count them. Adjust the density to 30,000 cells / well and inoculate 100 μL per well on a 96-well plate. Add 100 μL DMEM complete medium and incubate in a 37°C 5% CO2 incubator for 24 hours. Then discard the cell supernatant and wash twice with PBS buffer. Add the following reagents respectively:

[0071] Blank group: DMEM complete medium, 1% FBS;

[0072] Model group: DMEM complete medium, 1% FBS;

[0073] Positive group: DMEM complete medium, 1% FBS, 10 μmol / L Vc;

[0074] Drug administration group: DMEM complete medium, 1% FBS, and fish oil complex containing different concentrations of astaxanthin.

[0075] After culturing in a 37℃ 5% CO2 incubator for 24h, the cell supernatant was discarded and the cells were washed twice with PBS buffer. After DMEM complete medium was added to each group, the blank group was not treated, and 1.5mmol / L H2O2 was added to the model group, positive group and drug group, and the cells were cultured in a 37℃ 5% CO2 incubator for another 4h.

[0076] Then, various indicators were measured according to the instructions of the cell superoxide dismutase (SOD) activity assay kit, protein carbonyl content assay kit, malondialdehyde (MDA) assay kit, and reduced glutathione (GSH) assay kit.

[0077] (2) Experimental results

[0078] Table 1 is the SOD activity determination

[0079]

[0080]

[0081] Note: Compared with the blank control group, ## represents P<0.01, # represents P<0.05; compared with the model group, **** represents P<0.0001, *** represents P<0.001; ** represents P<0.01; * represents P<0.05.

[0082] As shown in Table 1, Examples 1-4 have more significant effects on improving the activity of cell SOD than Comparative Examples 1-4, while Comparative Examples 1 and 2 do not significantly improve the activity of cell SOD, which indicates that only astaxanthin or fish oil cannot achieve the desired effect. Only when astaxanthin and fish oil are mixed in a certain proportion can an unexpected effect be achieved. Figure 1 As shown, the cell SOD activity was dose-dependent to different concentrations of astaxanthin fish oil complex.

[0083] Table 2 shows the MDA content determination

[0084] Group MDA content (nmol / mgprot) Blank Group 0.314±0.035 Model Group 0.871±0.017## Positive group 0.409±0.041**** Example 1 - Low concentration dose group 0.530±0.025**** Example 1 - Medium concentration dose group 0.414±0.034**** Example 1 - High concentration dose group 0.432±0.041**** Example 2 - Medium concentration dose group 0.565±0.017**** Example 3 - Medium concentration dose group 0.534±0.023**** Example 4 - Medium concentration dose group 0.514±0.039**** Comparative Example 1-Low Concentration Dosage Group 0.784±0.034 Comparative Example 1-Medium concentration dose group 0.807±0.023 Comparative Example 1-High Concentration Dosage Group 0.795±0.049 Comparative Example 2-Low Concentration Dosage Group 0.754±0.029** Comparative Example 2-Medium Concentration Dose Group 0.667±0.046**** Comparative Example 2-High Concentration Dose Group 0.625±0.034**** Comparative Example 3-Medium Concentration Dose Group 0.651±0.059**** Comparative Example 4-Medium Concentration Dose Group 0.556±0.037****

[0085] Note: Compared with the blank control group, ## represents P<0.01, # represents P<0.05; compared with the model group, **** represents P<0.0001, *** represents P<0.001; ** represents P<0.01; * represents P<0.05.

[0086] As shown in Table 2 and Figure 2 As shown, the effects of Examples 1-4 are significant compared to Comparative Examples 1-4, and this phenomenon is particularly evident at medium and high concentrations. This proves that the combination of astaxanthin and fish oil can enhance the antioxidant effects of both.

[0087] Table 3 shows the determination of protein carbonyl content

[0088]

[0089]

[0090] Note: Compared with the blank control group, ## represents P<0.01, # represents P<0.05; compared with the model group, **** represents P<0.0001, *** represents P<0.001; ** represents P<0.01; * represents P<0.05.

[0091] Protein carbonylation refers to the oxidative damage of the amino acid residues on the side chains of proteins during oxidative stress and damage, which results in irreversible carbonylation damage. This carbonylation damage is the main sign of protein oxidative damage, and an increase in its content is usually associated with an increase in the degree of protein oxidative damage. Figure 3 As shown, after administration, the protein carbonyl content decreases with the increase of sample concentration, showing the dose dependence of astaxanthin, fish oil and astaxanthin fish oil complex. As shown in Table 3, the effect of the medium and high concentrations of the astaxanthin fish oil complex prepared in Example 1 on protein carbonyl is significantly enhanced compared with Comparative Examples 1-2, and this phenomenon is particularly obvious in the medium and high concentrations, which proves that the antioxidant effect of astaxanthin and fish oil can be enhanced after the combination.

[0092] Table 4 shows the determination of GSH content

[0093]

[0094]

[0095] Note: Compared with the blank control group, ## represents P<0.01, # represents P<0.05; compared with the model group,

[0096] **** represents P<0.0001, *** represents P<0.001; ** represents P<0.01; * represents P<0.05.

[0097] Reduced glutathione is one of the most abundant antioxidants in cells, and it maintains the balance between oxidation and antioxidation by neutralizing reactive oxygen species (ROS). When the production of ROS exceeds the neutralization capacity of the antioxidant system, oxidative stress (OS) may occur, and a decrease in GSH is considered a hallmark of OS. As shown in Table 4 and Figure 4 As shown, the GSH content in the experiment showed an obvious dose-dependent effect on Comparative Example 1, but the concentration of Comparative Example 2 had no obvious effect on the GSH content. Fish oil can regulate oxidative stress by regulating inflammation, oxidative stress and apoptosis, but it is not directly related to the GSH content, so the effect of the example on the GSH content also changes only by the change of astaxanthin concentration.

[0098] 2. Blood sugar lowering experiment

[0099] (1) Experimental methods:

[0100] The astaxanthin fish oil complexes prepared in Examples 1-4 and Comparative Examples 1-4 were ultrasonically mixed in 1 mL of DMSO, and then diluted with DMEM medium to the corresponding concentrations to obtain Example 1-low concentration dosage group, Example 1-medium concentration dosage group, Example 1-high concentration dosage group, Example 2-4-medium concentration dosage group, Comparative Example 1-low concentration dosage group, Comparative Example 1-medium concentration dosage group, Comparative Example 1-high concentration dosage group, Comparative Example 2-low concentration dosage group, Comparative Example 2-medium concentration dosage group, Comparative Example 2-high concentration dosage group, and Comparative Example 3-4 medium concentration dosage group.

[0101] Digest HepG2 cells to prepare cell suspension and count them. Adjust the density to 30,000 cells / well and inoculate 100 μL per well on a 96-well plate. Add 100 μL DMEM complete medium and incubate in a 37°C 5% CO2 incubator for 24 hours. Then discard the cell supernatant and wash twice with PBS buffer. Add the following reagents respectively:

[0102] Normal group: 25mmol / L DMEM complete medium, 1% FBS;

[0103] Model group: 30mmol / L DMEM complete medium, 1% FBS, 10 -7 mol / L insulin;

[0104] Positive group: 30mmol / L DMEM complete medium, 1% FBS, 10 -7 mol / L insulin, 10μmol / L rosiglitazone;

[0105] Drug group: 30mmol / L DMEM complete medium, 1% FBS, 10 -7 mol / L insulin, and fish oil complexes containing astaxanthin at different mass concentrations.

[0106] After culturing in a 37℃ 5% CO2 incubator for 24h, the cell supernatant was discarded and the cells were washed twice with PBS buffer. The normal group was added with DMEM high-glucose medium containing 25mmol / L glucose, and the model group, positive group and drug-treated group were added with DMEM high-glucose medium containing 30mmol / L respectively. The cells were placed in a 37℃ 5% CO2 incubator and continued to be cultured for 72h.

[0107] Then the cell supernatant was discarded and washed twice with PBS buffer. The normal group and the model group were added with DMEM high-glucose medium containing 25mmol / L glucose and DMEM high-glucose medium containing 30mmol / L, respectively. The positive group and the drug-treated group were added with high-glucose medium containing rosiglitazone and different concentrations of astaxanthin with 30mmol / L glucose content, respectively, and cultured in a CO2 incubator for 24h. The glucose content of the cell supernatant was determined according to the instructions of the cell glucose content determination kit.

[0108] (3) Experimental results

[0109] Table 5 shows the glucose consumption determination

[0110]

[0111]

[0112] Note: Compared with the blank control group, ## represents P<0.01, # represents P<0.05; compared with the model group, **** represents P<0.0001, *** represents P<0.001; ** represents P<0.01; * represents P<0.05.

[0113] As shown in Table 5 and Figure 5 As shown, it can be seen from the three concentrations of Example 1 that the blood sugar lowering effect of the medium concentration is significantly enhanced compared with the low concentration, and the blood sugar lowering effect of the high concentration is not significantly enhanced compared with the medium concentration; but the blood sugar lowering effect of all of them is much more significant than that of Comparative Examples 1-4.

[0114] In summary, the astaxanthin fish oil soft capsules provided by the present application and formulated in a specific ratio for assisting in lowering blood sugar have outstanding antioxidant activity and obvious blood sugar lowering effect, and have broad application prospects in diabetes.

Claims

1. An astaxanthin fish oil complex for assisting in lowering blood sugar, characterized in that: The invention comprises the following raw materials: astaxanthin oil and fish oil, wherein the mass ratio of the astaxanthin oil to the fish oil is 0.5-2:10-20.

2. The astaxanthin fish oil complex for assisting in lowering blood sugar according to claim 1, characterized in that: The mass ratio of the astaxanthin oil to the fish oil is 1:

12.

3. A method for preparing the astaxanthin fish oil complex for assisting in lowering blood sugar according to claim 1 or 2, characterized in that: The method comprises the following steps: fully stirring astaxanthin oil and fish oil to make them evenly mixed.

4. Use of the astaxanthin fish oil complex for assisting hypoglycemia according to claim 1 or 2 in the preparation of hypoglycemic drugs.

5. The use according to claim 4, characterized in that: The dosage form of the hypoglycemic drug is capsule, granule, tablet, oral solution or pill.

6. An astaxanthin fish oil capsule for assisting in lowering blood sugar, characterized in that: The contents include the astaxanthin fish oil complex for assisting in lowering blood sugar as described in claim 1 or 2 and vitamin E.

7. The astaxanthin fish oil capsule according to claim 6, characterized in that: The preparation method comprises the following steps: (1) Preparation of contents: Mix astaxanthin oil and fish oil, add vitamin E, and mix well; (2) Preparation of capsule skin material: Dissolve gelatin, glycerin and water and degas; (3) The contents and capsule skin are compressed to prepare astaxanthin fish oil soft capsules.

8. The astaxanthin fish oil capsule according to claim 7, characterized in that: The mass ratio of astaxanthin oil, fish oil and vitamin E in step (1) is 0.5-2:10-20:0.1-1.

9. The astaxanthin fish oil capsule according to claim 8, characterized in that: The mass ratio of astaxanthin oil, fish oil and vitamin E in step (1) is 1:12:0.

5.

10. The astaxanthin fish oil capsule according to claim 7, characterized in that: The volume ratio of gelatin, glycerol and water in step (2) is 1:0.4:1.

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