Synergistic anti-type 2 diabetes and anti-inflammatory composition and preparation method thereof

Abstract

The invention provides a synergistic anti-type 2 diabetes and anti-inflammatory composition and a preparation method thereof, and relates to the technical field of research and development of health care products / food additives. The raw materials of the composition are prepared by mixing low-molecular fucoidin, a mussel composite extract and an undaria pinnatifida extract, the mussel composite extract is prepared by the following steps: putting fresh mussels into saline water added with lycopene and caffeine, carrying out silt purification treatment, pulping, centrifuging, carrying out supercritical carbon dioxide extraction, and drying to obtain the mussel composite extract. Mixing tartaric acid and vitamin E, and freezing with liquid nitrogen; the undaria pinnatifida extract is obtained by grinding undaria pinnatifida into slurry, performing enzymolysis, performing microwave drying, performing subcritical water extraction and performing freeze-drying. The traditional Chinese medicine composition overcomes the defects in the prior art, can achieve the effect of low dosage and high curative effect by compounding multiple components, and achieves the synergistic effect of resisting type 2 diabetes and inflammation.

Description

Technical Field

[0001] This invention relates to the field of health product / food additive research and development technology, specifically to a composition and its preparation method that synergistically combats type 2 diabetes and inflammation. Background Technology

[0002] Type 2 diabetes is a common chronic metabolic disease, mainly caused by insufficient insulin secretion or insulin resistance, leading to elevated blood glucose levels. Treatment for type 2 diabetes typically involves different medications depending on the patient's specific condition. For example, biguanides such as metformin can increase glucose uptake and utilization by peripheral tissues and reduce hepatic glucose output; sulfonylureas can stimulate insulin secretion from pancreatic β-cells; meglitinides have a rapid onset and short duration of action, effectively lowering postprandial blood glucose; alpha-glucosidase inhibitors can delay carbohydrate digestion and absorption, lowering postprandial blood glucose; and thiazolidinediones can increase the sensitivity of target tissues to insulin, etc.

[0003] However, type 2 diabetes is often accompanied by oxidative stress, inflammation, and vascular complications. Existing drugs have limited effectiveness in combined intervention of these complications. Current anti-type 2 diabetes drugs or natural ingredients (such as chemical hypoglycemic agents and plant extracts) often have insufficient efficacy due to their single mechanism of action, or require high doses that may cause side effects. For example, traditional chemical hypoglycemic agents (such as metformin and sulfonylureas) may cause gastrointestinal reactions, hypoglycemia, or liver and kidney damage.

[0004] Existing functional food additives have limited effectiveness in treating type 2 diabetes and chronic inflammation. Current antidiabetic technologies face several challenges: 1) long-term use of chemical drugs (such as metformin and sulfonylureas) easily leads to drug resistance; 2) dietary and exercise adjustments suffer from poor adherence, making it difficult for patients to maintain treatment. Current anti-inflammatory technologies also present challenges: 1) long-term use of existing NSAIDs (such as ibuprofen) causes gastric mucosal damage (clinical reports show ≥30% of patients experiencing gastrointestinal adverse reactions), and long-term use of NSAIDs easily triggers gastrointestinal side effects; 2) glucocorticoids carry risks of osteoporosis and metabolic disorders; 3) natural anti-inflammatory components (such as curcumin and resveratrol) have low water solubility and first-pass effect, resulting in bioavailability of less than 5%, and there is a lack of synergistic natural compound formulations.

[0005] Existing studies have found that both fucoidan and mussel extract have good anti-inflammatory effects. However, common fucoidan (molecular weight > 100 kDa) has an intestinal absorption rate of <3% due to its large molecular weight. Mussel extract alone requires high doses (> 500 mg / day) to be effective, which is costly. Moreover, most mussel extracts are extracted directly from mussels using chemical solvents (such as ethanol), which is inefficient and leaves solvent residues. Existing technologies also use supercritical carbon dioxide to prepare mussel extracts, but the extracts are easily oxidized and the overall anti-inflammatory effect is poor.

[0006] Based on this, designing compound anti-inflammatory and anti-type 2 diabetes products to achieve high efficacy with low dosage is an important research direction at present. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a synergistic anti-type 2 diabetes and anti-inflammatory composition and its preparation method. The combination of multiple components can achieve high efficacy with low dosage, realizing the synergistic effect of anti-type 2 diabetes and anti-inflammatory.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A synergistic anti-type 2 diabetes and anti-inflammatory composition, wherein the raw materials of the composition include the following parts by weight: 3-5 parts of low molecular weight fucoidan, 3-5 parts of mussel compound extract, and 2-4 parts of wakame extract; the mussel compound extract is obtained by purifying fresh mussels in brine with added lycopene and caffeine, followed by pulping, centrifugation, supercritical carbon dioxide extraction, and then mixing with tartaric acid and vitamin E and freezing in liquid nitrogen; the wakame extract is obtained by grinding wakame into a pulp, enzymatically hydrolyzing, microwave drying, extracting with subcritical water, and then freeze-drying.

[0010] Preferably, the molecular weight of the low molecular weight fucoidan is ≤10kDa.

[0011] Preferably, the specific preparation method of the mussel compound extract includes the following steps:

[0012] S1-1. Freshly caught mussels are placed in water with a salinity of 25‰-35‰, a lycopene concentration of 40-60 mg / L, a caffeine concentration of 10-15 mg / L, and dissolved oxygen ≥5 mg / L for purification and cultivation for 5-7 days. After that, they are taken out and frozen to obtain pre-treated mussels for later use.

[0013] S1-2. Grind and pulp the pretreated mussels, centrifuge them, and extract the precipitate using supercritical carbon dioxide extraction to obtain the extract for later use.

[0014] S1-3. Add tartaric acid and vitamin E to the extract, mix well, and then freeze with liquid nitrogen to obtain mussel compound extract.

[0015] Preferably, the supercritical carbon dioxide extraction in step S1-2 is performed by placing 150g of precipitate in a supercritical fluid extraction device, delivering CO2 at a flow rate of 15kg / h for 2 hours, and controlling the temperature of the extract to be 70℃ and the pressure to be 50MPa.

[0016] Preferably, in steps S1-3, the amount of tartaric acid and vitamin E added is 0.01%-0.1% of the total mass of the extract.

[0017] Preferably, the method for preparing the wakame extract includes the following steps:

[0018] S2-1. After cleaning the wakame seaweed, grind it into a paste, add cellulase and pectinase and treat for 1-2 hours to obtain the enzymatic hydrolysate for later use.

[0019] S2-2. Place the enzymatic hydrolysate in a microwave oven and dry it to obtain microwaveable material for later use.

[0020] S2-3. Add an equal volume of water to the microwave material and place it in a high-pressure reactor. Adjust the pressure to 3-8 MPa and the temperature to 120-150℃, and perform subcritical water reaction treatment for 15-30 minutes. Then press, filter, and freeze dry to obtain wakame extract.

[0021] Preferably, in step S2-1, the amount of cellulase and pectinase added is 0.2%-0.3% of the total mass of wakame seaweed.

[0022] Preferably, the temperature of the drying process in the microwave oven in step S2-2 is controlled at 60-80℃, and the moisture content of the dried microwave material is controlled to be ≤12%.

[0023] The preparation method of the synergistic anti-type 2 diabetes and anti-inflammatory composition includes the following steps:

[0024] (1) Add the mussel compound extract to 10-20 times the volume of deionized water, sonicate and stir to homogenize, and then obtain the first mixture for later use.

[0025] (2) Mix low molecular weight fucoidan and wakame extract and add them to deionized water, then sonicate to obtain a second mixture for later use.

[0026] (3) After mixing the first mixture and the second mixture, the mixture is subjected to ultrasonic treatment under ice-water bath conditions to obtain a composite emulsion for later use.

[0027] (4) The composite emulsion was freeze-dried to obtain a composition with synergistic anti-type 2 diabetes and anti-inflammatory effects.

[0028] This invention provides a synergistic anti-type 2 diabetes and anti-inflammatory composition and its preparation method, which has the following advantages compared with the prior art:

[0029] This invention uses a combination of low-molecular-weight fucoidan, mussel extract, and wakame extract as active ingredients, which can effectively regulate blood sugar and reduce inflammation. Furthermore, the mussels are purified and treated with lycopene and caffeine before the extract is prepared, effectively enhancing the activity of the subsequent mussel extract. The wakame, extracted with subcritical water, enzymatically hydrolyzed, and microwave-dried, combined with the low-molecular-weight fucoidan and mussel extract, overcomes the strong dose-dependency of traditional anti-inflammatory agents and further enhances the anti-type 2 diabetes effect. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1:

[0032] Raw material acquisition:

[0033] 1. Obtaining low molecular weight fucoidan:

[0034] The seaweed is ultra-finely pulverized and extracted with water. The extract is then circulated to dissolve other ultra-finely pulverized seaweed until a saturated filtrate is produced. The pH of the filtrate is adjusted to 4 with citric acid, and then filtered through a microfiltration membrane (the microfiltration membrane is selected from ceramic microfiltration membranes with a pore size of 0.5 micrometers, and the flow rate of the filtrate through the ceramic microfiltration membrane is 15-200 L / h) to obtain the first filtrate.

[0035] Calcium chloride was added to the first filtrate to make the calcium chloride content 1.5% for sedimentation, and then centrifuged at 14000g using a disc centrifuge to obtain algin and supernatant.

[0036] The supernatant was filtered at a flow rate of 20 L / h using an 800 Dalton polyethersulfone (PES) composite membrane (the polyethersulfone composite membrane can effectively retain large molecules in the supernatant while allowing small molecules such as polysaccharides, small impurities such as salts, inorganic substances and free heavy metals to pass through).

[0037] Collect the small molecule filtrate, add ethanol to make the ethanol concentration of the filtrate reach 80%, precipitate the polysaccharide with alcohol, collect the precipitate, and spray dry it to obtain small molecule fucoidan powder.

[0038] 2. Mussel compound extract:

[0039] (1) Freshly caught mussels were placed in water with a salt concentration of 25‰-35‰ and dissolved oxygen ≥5mg / L for purification and aquaculture treatment for 7 days. During the purification and aquaculture treatment, a certain concentration of lycopene and caffeine were added to the water. After treatment, the mussels were directly taken out and frozen at -18℃ for storage.

[0040] (2) After thawing the frozen mussels, remove the shells and grind them into a pulp. Then, centrifuge at 4000 r / min for 10 min to remove the clear liquid and retain the precipitate.

[0041] (3) Take 150g of the above precipitate and place it in a supercritical fluid extraction device. Flow CO2 at a flow rate of 15kg / h for 2 hours. Control the temperature of the extract to 70℃ and the pressure to 50MPa for extraction. Obtain the extract (which is amber-colored lipid). Add 0.05% of the total mass of the extract, tartaric acid and vitamin E to the extract and mix well to obtain mussel compound extract. Store in liquid nitrogen at -80℃ for later use.

[0042] Following the preparation method described above, and controlling the concentrations of lycopene and caffeine in the water during mussel purification and aquaculture according to Table 1 below, different mussel compound extracts were obtained:

[0043] Table 1

[0044] Mussel compound extract Lycopene concentration (mg / L) Caffeine concentration (mg / L) GLM-1 0 0 GLM-2 20 5 GLM-3 40 10 GLM-4 60 15 GLM-5 80 20

[0045] 3. Preparation of wakame extract:

[0046] 3.1 Preparation of WK-1:

[0047] (1) After cleaning the wakame seaweed, grind it into a paste, add 0.2% of the total mass of wakame seaweed cellulase and pectinase and treat for 1.5 hours to obtain the enzymatic hydrolysate for later use;

[0048] (2) Place the enzymatic hydrolysate in a microwave oven and dry it at 70°C until the moisture content is ≤12% to obtain microwave material for later use.

[0049] (3) After adding an equal volume of water to the microwave material, place it in a high-pressure reactor, adjust the pressure to 5 MPa and the temperature to 130°C, and carry out subcritical water reaction treatment for 25 min. Then press, filter and freeze dry to obtain the wakame extract as WK-1.

[0050] 3.2 Preparation of WK-2:

[0051] (1) After cleaning the wakame seaweed, grind it into a paste, add 0.2% of the total mass of wakame seaweed cellulase and pectinase and treat for 1.5 hours to obtain the enzymatic hydrolysate for later use;

[0052] (2) Add an equal volume of water to the enzymatic hydrolysate and place it in a high-pressure reactor. Adjust the pressure to 5 MPa and the temperature to 130°C. Perform a subcritical water reaction for 25 min. Then press, filter, and freeze dry to obtain the wakame extract WK-2.

[0053] 3.3 Preparation of WK-3:

[0054] (1) After cleaning the wakame seaweed, grind it into a paste, add 0.2% of the total mass of wakame seaweed cellulase and pectinase and treat for 1.5 hours to obtain the enzymatic hydrolysate for later use;

[0055] (2) Place the enzymatic hydrolysate in a microwave oven and dry it at 70°C until the moisture content is ≤12% to obtain microwave material for later use.

[0056] (3) After adding an equal volume of water to the microwave material, it was extracted by ultrasonic extraction at 400W for 25 minutes at a water bath temperature of 60℃. Then it was pressed, filtered and freeze-dried to obtain the WK-3 extract of wakame seaweed.

[0057] Example 2:

[0058] Preparation of the composition:

[0059] (1) Add the mussel compound extract to 15 times the volume of deionized water and treat it under 600W ultrasound for 10 min. Then stir and homogenize it at 1200r / min for 10 min to obtain the first mixture for later use.

[0060] (2) Mix low molecular weight fucoidan and wakame extract and add them to 5 times the total mass of low molecular weight fucoidan and wakame extract in deionized water. Sonicate at 600W for 15 minutes to obtain a second mixture for later use.

[0061] (3) After mixing the first mixture and the second mixture, place them in an ice-water bath and sonicate at 600W for 10 minutes to obtain a composite emulsion. Freeze-dry the emulsion to obtain the composition.

[0062] Following the preparation method described above, different compositions were prepared according to the formulations in Table 2 below, based on the respective weight parts:

[0063] Table 2

[0064] Group Formula (parts by weight) Experimental group 1 4 parts low molecular weight fucoidan + 4 parts GLM-1 + 3 parts WK-1 Experimental group 2 4 parts low molecular weight fucoidan + 4 parts GLM-2 + 3 parts WK-1 Experimental group 3 4 parts low molecular weight fucoidan + 4 parts GLM-3 + 3 parts WK-1 Experimental group 4 4 parts low molecular weight fucoidan + 4 parts GLM-4 + 3 parts WK-1 Experimental group 5 4 parts low molecular weight fucoidan + 4 parts GLM-5 + 3 parts WK-1 Experimental group 6 4 parts low molecular weight fucoidan + 4 parts GLM-3 + 3 parts WK-2 Experimental group 7 4 parts low molecular weight fucoidan + 4 parts GLM-3 + 3 parts WK-3 Experimental group 8 4 parts low molecular weight fucoidan + 4 parts GLM-3

[0065] Example 3:

[0066] The anti-inflammatory and anti-type 2 diabetes effects of the above experimental groups were tested:

[0067] 1. Anti-inflammatory effect test:

[0068] (1) Cell Culture

[0069] After resuscitation, RAW 264.7 cells were added to 5 mL of DMEM medium containing 10% FBS, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cells were resuspended in the medium, seeded into culture flasks, and incubated at 37°C in a 5% CO2 incubator.

[0070] When the cell density reaches 80%–90%, passage the cells. Discard the old culture medium, wash the cells twice with PBS, digest them with trypsin, observe them under an inverted microscope, and stop digestion by adding culture medium containing 10% FBS after the cells become rounded and detached. Pipette the cells to make a single-cell suspension, and seed them into new culture flasks at a ratio of 1:3 for continued culture.

[0071] Cells in the logarithmic growth phase were harvested, digested with trypsin, and counted. The cell density was adjusted to 5 × 10⁶ cells / year using DMEM medium containing 10% FBS. 4 Cells were seeded at a density of 100 μL / mL into 96-well plates and incubated overnight to allow the cells to adhere.

[0072] (2) LPS induction

[0073] After cell adhesion, the culture medium was replaced with low serum (1% FBS) medium for synchronization treatment for 23 h. Then, 1 μg / mL of LPS was added, and the cells were cultured for another 2 h.

[0074] (3) Drug effects:

[0075] After 2 hours of incubation, experimental groups 1-9, GLM-3, and low-molecular-weight fucoidan (5, 25, and 50 μg / mL, respectively) were added to the culture medium (one group served as a control without additional components, acting as the LPS treatment group). Incubation continued for 12 hours. The concentrations of IL-6 and TNF-α in the culture supernatant were detected using an enzyme-linked immunosorbent assay (ELISA) with a mouse IL-6 detection kit and a mouse TNF-α ELISA kit. The specific results are shown in Tables 4 and 5 below.

[0076] Table 4. Levels of variation in IL-6 (with LPS-treated group data as 100%)

[0077]

[0078] Table 5. Changes in TNF-α levels (with LPS-treated group data as 100%)

[0079]

[0080] The above tests show that the compound drug compositions prepared in experimental groups 3 and 4 can significantly reduce the levels of TNF-α and IL-6, thereby inhibiting inflammation.

[0081] 2. Glucose uptake by muscle cells C2C12

[0082] (1) Mouse myoblast cell line C2C12 was seeded in DMEM medium containing 10% fetal bovine serum and cultured in an incubator containing 5% CO2 at 37°C, with the medium changed every other day. When the cells reached 70%–80% confluence, they were passaged at a ratio of 1:4.

[0083] (2) When the C2C12 cells reach approximately 80% confluence, they are passaged and seeded into intervention plates. When the cells grow to 80%–90% confluence, the medium is changed to differentiation medium (DMEM medium containing 2% horse serum) to induce differentiation, and the medium is changed every 24 hours. After 7 days of culture, more than 90% of the cells will become mature skeletal muscle cells for subsequent experiments;

[0084] (3) Differentiated C2C12 cells (1×10⁶ cells per well) 5 Each sample was placed in a 24-well plate and pre-incubated for 24 hours in DMEM medium containing 10% fetal bovine serum (FBS).

[0085] On the day of the experiment, cells were placed in serum-free DMEM medium, and the drugs for experimental groups 1-8 were added at concentrations of 5, 25, 50, and 100 μg / ml, respectively. A control group containing 10 μg / ml insulin was set up, and an untreated group was set up without any drugs or insulin. After incubation for 24 hours, 50 μl of 1 mM 2DG solution was added to each well. The reaction was terminated by adding 25 μl of stop buffer, followed by 25 μl of neutralization buffer. Then, 100 μl of 2DG6P assay reagent was added to each well, and after shaking to mix, the cells were incubated at room temperature for 1 hour. Detection was performed using a Tecan Trading AG SPARK 10M chemiluminescence analyzer.

[0086] The specific results are shown below. The cellular glucose uptake in the untreated group was recorded as 100%. Changes in cellular glucose uptake were detected in each group. The control group containing 10 μg / ml insulin showed a 149.1% change in cellular glucose uptake.

[0087] Table 6. Changes in cellular glucose uptake due to different drug additions.

[0088]

[0089] The above tests show that when the drugs in experimental groups 3 and 4 are used at a dosage of 25 μg / mL, their effects are close to those of the control group with added insulin. Moreover, compared with the use of low molecular weight fucoidan and GLM-3 alone, they can synergistically promote the increase of cellular glucose uptake at low doses.

[0090] 3. α-Amylase inhibition experiment:

[0091] The enzyme inhibition kit (catalog number ab283391, Abogen Biosciences, Cambridge, UK) was used according to the manufacturer's instructions, and experiments were conducted in 96-well plates. Each reagent was first diluted 100-fold, and then diluted 3-fold with assay buffer. 50 μL of each reagent was mixed with 100 μL of α-amylase-substrate reaction mixture to prepare final concentration gradients of 25, 50, 100, and 500 μg / mL. The optical density was measured at 405 nm in kinetic mode at room temperature, with data read every 5 minutes. An inhibitor control group provided by the manufacturer was used for comparison (based on the inhibitor control IC50 (88.5%) provided by the kit).

[0092] The specific results are shown in Table 7 below:

[0093] Table 7

[0094]

[0095] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A synergistic anti-type 2 diabetes and anti-inflammatory composition, characterized in that, The raw materials of the composition include the following parts by weight: 3-5 parts of low molecular weight fucoidan, 3-5 parts of mussel compound extract, and 2-4 parts of wakame extract; The mussel compound extract is obtained by placing fresh mussels in a brine solution containing lycopene and caffeine for mud and sand purification, followed by pulping, centrifugation, supercritical carbon dioxide extraction, and then mixing with tartaric acid and vitamin E and freezing with liquid nitrogen. The wakame extract was obtained by grinding wakame into a paste, enzymatically hydrolyzing it, microwaving it, extracting it with subcritical water, and then freeze-drying it.

2. The composition according to claim 1, characterized in that: The molecular weight of the low molecular weight fucoidan is ≤10kDa.

3. The composition according to claim 1, characterized in that, The specific preparation method of the mussel compound extract includes the following steps: S1-1. Freshly caught mussels are placed in water with a salinity of 25‰-35‰, a lycopene concentration of 40-60 mg / L, a caffeine concentration of 10-15 mg / L, and dissolved oxygen ≥5 mg / L for purification and cultivation for 5-7 days. After that, they are taken out and frozen to obtain pre-treated mussels for later use. S1-2. Grind and pulp the pretreated mussels, centrifuge them, and extract the precipitate using supercritical carbon dioxide extraction to obtain the extract for later use. S1-3. Add tartaric acid and vitamin E to the extract, mix well, and then freeze with liquid nitrogen to obtain mussel compound extract.

4. The composition according to claim 3, characterized in that: In step S1-2, the supercritical carbon dioxide extraction method involves placing 150g of precipitate in a supercritical fluid extraction device, delivering CO2 at a flow rate of 15kg / h for 2 hours, and controlling the temperature of the extract at 70℃ and the pressure at 50MPa.

5. The composition according to claim 3, characterized in that: In steps S1-3, the amount of tartaric acid and vitamin E added is 0.01%-0.1% of the total mass of the extract.

6. The composition according to claim 1, characterized in that, The preparation method of the wakame extract includes the following steps: S2-1. After cleaning the wakame seaweed, grind it into a paste, add cellulase and pectinase and treat for 1-2 hours to obtain the enzymatic hydrolysate for later use. S2-2. Place the enzymatic hydrolysate in a microwave oven and dry it to obtain microwaveable material for later use. S2-3. Add an equal volume of water to the microwave material and place it in a high-pressure reactor. Adjust the pressure to 3-8 MPa and the temperature to 120-150℃, and perform subcritical water reaction treatment for 15-30 minutes. Then press, filter, and freeze dry to obtain wakame extract.

7. The composition according to claim 6, characterized in that: In step S2-1, the amount of cellulase and pectinase added is 0.2%-0.3% of the total mass of wakame seaweed.

8. The composition according to claim 6, characterized in that: In step S2-2, the temperature of the drying process in the microwave oven is controlled at 60-80℃, and the moisture content of the dried microwave material is controlled to be ≤12%.

9. A method for preparing the composition according to any one of claims 1-8, characterized in that: The preparation method includes the following steps: (1) Add the mussel compound extract to 10-20 times the volume of deionized water, sonicate and stir to homogenize, and then obtain the first mixture for later use. (2) Mix low molecular weight fucoidan and wakame extract and add them to deionized water, then sonicate to obtain a second mixture for later use. (3) After mixing the first mixture and the second mixture, the mixture is subjected to ultrasonic treatment under ice-water bath conditions to obtain a composite emulsion for later use. (4) The composite emulsion was freeze-dried to obtain a composition with synergistic anti-type 2 diabetes and anti-inflammatory effects.

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