Use of pomelo powder or pomelo juice targeting GLP-1 receptor in preparation of products for weight loss and improvement of blood glucose and blood lipid disorders

Abstract

The present application relates to the application of pomelo powder or pomelo juice targeting GLP-1 receptor in the preparation of weight loss and blood glucose and blood lipid disorder improvement products, wherein the pomelo powder or pomelo juice is obtained by a preparation method comprising the following steps: reflux extraction after ultrasonic-assisted enzymatic hydrolysis of pomelo raw materials, and concentration of the filtrate after extraction, separation and purification to obtain pomelo powder or pomelo juice. The present application creatively uses pomelo peel and other tissues as raw materials, converts agricultural waste that is discarded or used at low value into high-value medical and functional food raw materials, and for the first time verifies that pomelo powder or pomelo juice can be used as a natural GLP-1R receptor agonist and can play an excellent role in reducing blood sugar and blood lipid and protecting the cardiovascular system. The present application creatively designs a specific extraction method for pomelo powder or pomelo juice, which can achieve excellent extraction effect and has strong practicability and adaptability.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of grapefruit powder or grapefruit juice that targets the GLP-1 receptor in the preparation of products for weight loss and improvement of blood sugar and lipid disorders. Background Technology

[0002] Metabolic diseases, such as type 2 diabetes, obesity, and non-alcoholic fatty liver disease, have become major global public health problems. These diseases are often accompanied by pathological states such as abnormal blood glucose regulation and lipid metabolism disorders, significantly increasing the risk of cardiovascular disease. Glucagon-like peptide-1 (GLP-1) is an insulin-stimulating hormone secreted by intestinal L cells. By activating the glucagon-like peptide-1 receptor (GLP-1R), it promotes insulin secretion, inhibits glucagon release, delays gastric emptying, and suppresses appetite, making it a popular target for the treatment of obesity and type 2 diabetes. Currently, several GLP-1R agonists, such as liraglutide and semaglutide, have been developed and have shown significant benefits in lowering blood glucose, weight loss, and cardiovascular protection. However, these drugs are mostly injectable peptide formulations, which have limitations such as high production costs, the need for injection administration, and the potential for gastrointestinal adverse reactions. Natural products, as a valuable resource for GLP-1R agonists, are increasingly demonstrating their value. Research on GLP-1R agonists is expanding from chemically synthesized drugs to natural product sources.

[0003] In the prior art, patent publication number CN119614319A discloses that pomelo peel extract has antioxidant activity, and literature publication number CN110558469A reports the hypoglycemic effect of pomelo extract. However, the above-mentioned prior art does not involve the activation of GLP-1R, nor does it reveal the relationship between its hypoglycemic and lipid-lowering activities and GLP-1R agonism. Furthermore, existing GLP-1R agonists such as Liraglutide and Semaglutide are chemically synthesized polypeptide drugs, which have drawbacks such as injection administration, high production costs, and significant gastrointestinal adverse reactions. No naturally derived, orally effective GLP-1R agonists have been disclosed.

[0004] Therefore, it is particularly urgent to develop a product that is naturally derived, inexpensive, easy to use, and has GLP-1R agonist activity to meet the market demand for the prevention and adjuvant treatment of metabolic diseases. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an application of grapefruit powder or grapefruit juice targeting the GLP-1 receptor in the preparation of products for weight loss and improvement of blood sugar and lipid disorders.

[0006] To achieve this objective, the present invention employs the following technical solution: In a first aspect, the present invention provides the application of grapefruit powder or grapefruit juice targeting the GLP-1 receptor in the preparation of products for weight loss and improvement of blood sugar and lipid disorders, wherein the grapefruit powder or grapefruit juice is obtained by a preparation method comprising the following steps: After ultrasonic-assisted enzymatic hydrolysis, the pomelo raw material is refluxed for extraction. After extraction, the filtrate is concentrated and purified to obtain pomelo powder or pomelo juice.

[0007] This invention creatively utilizes pomelo peel and other tissues as raw materials, transforming agricultural waste that was previously discarded or underutilized into high-value pharmaceutical and functional food ingredients. It is the first to verify that pomelo powder or juice can act as a natural GLP-1 receptor agonist, exhibiting excellent hypoglycemic, lipid-lowering, and cardiovascular protective effects. This invention also creatively designs a specific extraction method for pomelo powder or juice, achieving excellent extraction results and demonstrating strong practicality and adaptability.

[0008] Preferably, the pomelo raw material includes any one or a combination of at least two of pomelo peel, pomelo pulp, and pomelo seeds.

[0009] Preferably, the pomelo raw material is selected from a combination of pomelo peel and pomelo pulp.

[0010] Extracting pomelo peel and pulp together as raw materials can achieve greater extraction efficiency and better separation of active ingredients from pomelo.

[0011] Preferably, the solvent used for the ultrasound-assisted enzymatic hydrolysis is an ethanol-water solution with a volume fraction of 30-50% (e.g., 30%, 35%, 40%, 45%, 50%, etc.).

[0012] Preferably, during the ultrasound-assisted enzymatic hydrolysis, the ratio of pomelo raw material to solvent is 1:(8-20) g / mL (for example, it can be 1:8 g / mL, 1:10 g / mL, 1:12 g / mL, 1:14 g / mL, 1:16 g / mL, 1:20 g / mL, etc.).

[0013] Preferably, the ultrasonic power of the ultrasound-assisted enzymatic hydrolysis is 200-400 W (e.g., 200 W, 230 W, 260 W, 290 W, 320 W, 350 W, 380 W, 400 W, etc.).

[0014] Preferably, the ultrasound-assisted enzymatic hydrolysis uses a complex enzyme of cellulase, pectinase, and β-glucosidase in a mass ratio of (0.5-3):(0.5-2):1 (where the specific value of 0.5-3 can be 0.5, 1, 1.5, 2, 2.5, 3, etc.; and the specific value of 0.5-2 can be 0.5, 1, 1.5, 2, etc.).

[0015] Preferably, the compound enzyme accounts for 3-6% of the mass of the pomelo raw material (e.g., 3%, 4%, 5%, 6%, etc.).

[0016] Preferably, the temperature of the ultrasound-assisted enzymatic hydrolysis is 40-55℃ (e.g., 40℃, 42℃, 44℃, 46℃, 48℃, 50℃, 52℃, 55℃, etc.), and the time is 30-60 min (e.g., 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.).

[0017] Preferably, the reflux extraction is performed by adding anhydrous ethanol or an aqueous ethanol solution to the system until the final concentration of ethanol is 55-70% (e.g., 55%, 57%, 60%, 62%, 65%, 67%, 70%, etc.).

[0018] Preferably, the reflux extraction time is 1-3 h (e.g., 1 h, 1.5 h, 2 h, 2.5 h, 3 h, etc.).

[0019] Preferably, the separation and purification method includes macroporous adsorption resin column chromatography.

[0020] Preferably, the macroporous adsorption resin includes any one or a combination of at least two of the following types: D101, AB-8, HPD-100, and XAD-7.

[0021] Preferably, the macroporous adsorption resin is selected from a combination of D101 and AB-8 types.

[0022] In a second aspect, the present invention provides the use of pomelo powder or pomelo juice in the preparation of GLP-1 receptor agonists.

[0023] Preferably, the pomelo powder or pomelo juice is obtained by a preparation method comprising the following steps: After ultrasonic-assisted enzymatic hydrolysis, the pomelo raw material is refluxed for extraction. After extraction, the filtrate is concentrated and purified to obtain pomelo powder or pomelo juice.

[0024] Preferably, the pomelo raw material includes any one or a combination of at least two of pomelo peel, pomelo pulp, and pomelo seeds.

[0025] Preferably, the pomelo raw material is selected from a combination of pomelo peel and pomelo pulp.

[0026] Preferably, the solvent used for the ultrasound-assisted enzymatic hydrolysis is an ethanol-water solution with a volume fraction of 30-50% (e.g., 30%, 35%, 40%, 45%, 50%, etc.).

[0027] Preferably, during the ultrasound-assisted enzymatic hydrolysis, the ratio of pomelo raw material to solvent is 1:(8-20) g / mL (for example, it can be 1:8 g / mL, 1:10 g / mL, 1:12 g / mL, 1:14 g / mL, 1:16 g / mL, 1:20 g / mL, etc.).

[0028] Preferably, the ultrasonic power of the ultrasound-assisted enzymatic hydrolysis is 200-400 W (e.g., 200 W, 230 W, 260 W, 290 W, 320 W, 350 W, 380 W, 400 W, etc.).

[0029] Preferably, the ultrasound-assisted enzymatic hydrolysis uses a complex enzyme of cellulase, pectinase, and β-glucosidase in a mass ratio of (0.5-3):(0.5-2):1 (where the specific value of 0.5-3 can be 0.5, 1, 1.5, 2, 2.5, 3, etc.; and the specific value of 0.5-2 can be 0.5, 1, 1.5, 2, etc.).

[0030] Preferably, the compound enzyme accounts for 3-6% of the mass of the pomelo raw material (e.g., 3%, 4%, 5%, 6%, etc.).

[0031] Preferably, the temperature of the ultrasound-assisted enzymatic hydrolysis is 40-55℃ (e.g., 40℃, 42℃, 44℃, 46℃, 48℃, 50℃, 52℃, 55℃, etc.), and the time is 30-60 min (e.g., 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.).

[0032] Preferably, the reflux extraction is performed by adding anhydrous ethanol or an aqueous ethanol solution to the system until the final concentration of ethanol is 55-70% (e.g., 55%, 57%, 60%, 62%, 65%, 67%, 70%, etc.).

[0033] Preferably, the reflux extraction time is 1-3 h (e.g., 1 h, 1.5 h, 2 h, 2.5 h, 3 h, etc.).

[0034] Preferably, the separation and purification method includes macroporous adsorption resin column chromatography.

[0035] Preferably, the macroporous adsorption resin includes any one or a combination of at least two of the following types: D101, AB-8, HPD-100, and XAD-7.

[0036] Preferably, the macroporous adsorption resin is selected from a combination of D101 and AB-8 types.

[0037] Thirdly, the present invention provides the use of pomelo powder or pomelo juice in the preparation of products for the prevention or treatment of metabolic diseases, wherein the pomelo powder or pomelo juice is obtained by a preparation method comprising the following steps: After ultrasonic-assisted enzymatic hydrolysis, the pomelo raw material is refluxed for extraction. After extraction, the filtrate is concentrated and purified to obtain pomelo powder or pomelo juice.

[0038] Preferably, the metabolic disease includes any one of obesity, atherosclerosis, hyperlipidemia, non-alcoholic fatty liver disease, and metabolic syndrome.

[0039] Preferably, the pomelo raw material includes any one or a combination of at least two of pomelo peel, pomelo pulp, and pomelo seeds.

[0040] Preferably, the pomelo raw material is selected from a combination of pomelo peel and pomelo pulp.

[0041] Preferably, the solvent used for the ultrasound-assisted enzymatic hydrolysis is an ethanol-water solution with a volume fraction of 30-50% (e.g., 30%, 35%, 40%, 45%, 50%, etc.).

[0042] Preferably, during the ultrasound-assisted enzymatic hydrolysis, the ratio of pomelo raw material to solvent is 1:(8-20) g / mL (for example, it can be 1:8 g / mL, 1:10 g / mL, 1:12 g / mL, 1:14 g / mL, 1:16 g / mL, 1:20 g / mL, etc.).

[0043] Preferably, the ultrasonic power of the ultrasound-assisted enzymatic hydrolysis is 200-400 W (e.g., 200 W, 230 W, 260 W, 290 W, 320 W, 350 W, 380 W, 400 W, etc.).

[0044] Preferably, the ultrasound-assisted enzymatic hydrolysis uses a complex enzyme of cellulase, pectinase, and β-glucosidase in a mass ratio of (0.5-3):(0.5-2):1 (where the specific value of 0.5-3 can be 0.5, 1, 1.5, 2, 2.5, 3, etc.; and the specific value of 0.5-2 can be 0.5, 1, 1.5, 2, etc.).

[0045] Preferably, the compound enzyme accounts for 3-6% of the mass of the pomelo raw material (e.g., 3%, 4%, 5%, 6%, etc.).

[0046] Preferably, the temperature of the ultrasound-assisted enzymatic hydrolysis is 40-55℃ (e.g., 40℃, 42℃, 44℃, 46℃, 48℃, 50℃, 52℃, 55℃, etc.), and the time is 30-60 min (e.g., 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.).

[0047] Preferably, the reflux extraction is performed by adding anhydrous ethanol or an aqueous ethanol solution to the system until the final concentration of ethanol is 55-70% (e.g., 55%, 57%, 60%, 62%, 65%, 67%, 70%, etc.).

[0048] Preferably, the reflux extraction time is 1-3 h (e.g., 1 h, 1.5 h, 2 h, 2.5 h, 3 h, etc.).

[0049] Preferably, the separation and purification method includes macroporous adsorption resin column chromatography.

[0050] Preferably, the macroporous adsorption resin includes any one or a combination of at least two of the following types: D101, AB-8, HPD-100, and XAD-7.

[0051] Preferably, the macroporous adsorption resin is selected from a combination of D101 and AB-8 types.

[0052] Preferably, the product for the prevention or treatment of metabolic diseases further includes pharmaceutically acceptable excipients.

[0053] Preferably, the excipients include any one or a combination of at least two of the following: carrier, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0054] Preferably, the pomelo powder or pomelo juice in the product for the prevention or treatment of metabolic diseases has a mass fraction of 5-50% (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, etc.).

[0055] All other specific point values ​​not listed above within the numerical ranges mentioned above can be selected and are all within the protection scope of this invention. For the sake of brevity, they will not be described in detail here.

[0056] Compared with the prior art, the present invention has the following beneficial effects: This invention creatively utilizes pomelo peel and other tissues as raw materials, transforming agricultural waste that was previously discarded or underutilized into high-value pharmaceutical and functional food ingredients. It is the first to verify that pomelo powder or juice can act as a natural GLP-1 receptor agonist, exhibiting excellent hypoglycemic, lipid-lowering, and cardiovascular protective effects. This invention also creatively designs a specific extraction method for pomelo powder or juice, achieving excellent extraction results and demonstrating strong practicality and adaptability. Attached Figure Description

[0057] Figure 1 The high-performance liquid chromatogram of pomelo powder in Example 1 is shown below. Figure 2 The mass spectrum of senna-based active ingredient senna-based glycoside in pomelo powder from Example 1 is shown. Figure 3 The mass spectrum of the active ingredient, neo-North American sage glycoside, in the pomelo powder of Example 1; Figure 4 The mass spectrum of neohesperidin, the active ingredient in pomelo powder from Example 1; Figure 5 The mass spectrum of naringin, the active ingredient in pomelo powder from Example 1, is shown. Figure 6 The mass spectrum of rutin, an active ingredient in pomelo powder from Example 1, is shown. Figure 7 Fluorescence map of plasmid transfection for constructing a high-throughput screening model targeting GLP-1R (left) and GLP-1R protein expression map of a single stable transgenic strain (right). Figure 8 The effects of pomelo powder obtained in Examples 1-2 and Comparative Examples 1-2 on cell viability; Figure 9 The graph shows the changes in body weight of mice in the WT group, CTL group, HFD group, Examples 1-2, and Comparative Examples 1-2. Figure 10 Blood lipid statistics for mice in the WT group, CTL group, HFD group, Examples 1-2, and Comparative Examples 1-2; Figure 11 Blood glucose statistics for mice in the WT group, CTL group, HFD group, Examples 1-2, and Comparative Examples 1-2; Figure 12 The images show pathological staining of liver, white fat cells, and aorta in mice from the WT group, CTL group, HFD group, and Example 1 group. Figure 13 The graph shows the relative expression levels of intestinal-related proteins in mice from the WT group, CTL group, HFD group, and Example 1 group. Detailed Implementation

[0058] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0059] Example 1 This embodiment provides a pomelo powder, and the preparation method is as follows: (1) The peel and pulp of pomelo were used as raw materials and mixed with 40% ethanol-water solution at a material-liquid ratio of 1:15. The mixture was subjected to ultrasonic-assisted enzymatic hydrolysis at 45°C with an ultrasonic power of 300 W. A complex enzyme consisting of cellulase, pectinase and β-glucosidase in a mass ratio of 2:1:1 was used for enzymatic hydrolysis. The complex enzyme accounted for 4% of the mass of the pomelo raw material. (2) After the enzymatic hydrolysis, anhydrous ethanol was added to the system until the final concentration of ethanol was 70% for reflux extraction. The extraction time was 2.5 h. After the extraction, the filtrate was concentrated under reduced pressure to remove alcohol. Then, water was added to dilute the concentrate, and the solution was filtered. The solution was passed through a mixed macroporous resin of type D101 and type AB-8 and eluted with 50% ethanol as the eluent. Then, the solution was concentrated under reduced pressure and spray-dried to obtain pomelo powder.

[0060] Example 2 This embodiment provides a pomelo powder, and the preparation method is as follows: (1) The peel and pulp of pomelo were used as raw materials and mixed with 40% ethanol-water solution at a material-liquid ratio of 1:15. The mixture was subjected to ultrasonic-assisted enzymatic hydrolysis at 45°C with an ultrasonic power of 300 W. A complex enzyme consisting of cellulase, pectinase and β-glucosidase in a mass ratio of 2:1:1 was used for enzymatic hydrolysis. The complex enzyme accounted for 4% of the mass of the pomelo raw material. (2) After the enzymatic hydrolysis, anhydrous ethanol was added to the system until the final concentration of ethanol was 70% for reflux extraction. The extraction time was 2.5 h. After the extraction, the filtrate was concentrated under reduced pressure to remove alcohol. Then, water was added to dilute the concentrate, and the solution was filtered and passed through a D101 macroporous resin column. 50% ethanol was used as the eluent for elution. Then, the solution was concentrated under reduced pressure and spray-dried to obtain pomelo powder.

[0061] Comparative Example 1 This comparative example provides a pomelo powder, and the preparation method is as follows: (1) The peel and pulp of pomelo were used as raw materials and mixed with 50% ethanol-water solution at a material-liquid ratio of 1:15. The mixture was subjected to ultrasonic-assisted enzymatic hydrolysis at 50°C with an ultrasonic power of 500 W. A complex enzyme consisting of cellulase, pectinase and β-glucosidase in a mass ratio of 3:1.5:1 was used for enzymatic hydrolysis. The complex enzyme accounted for 6% of the mass of the pomelo raw material. (2) After extraction, the filtrate was concentrated under reduced pressure to remove alcohol, then water was added to dilute the concentrate, filtered, and passed through a mixed macroporous resin of type D101 and type AB-8. 50% ethanol was used as the eluent for elution, followed by reduced pressure concentration and spray drying to obtain pomelo powder.

[0062] Comparative Example 2 This comparative example provides a pomelo powder, and the preparation method is as follows: The peel and pulp of pomelo were used as raw materials and mixed with an 80% ethanol-water solution at a material-liquid ratio of 1:12. The mixture was then refluxed for extraction for 4 hours. After extraction, the filtrate was concentrated under reduced pressure to remove the alcohol. The concentrate was then diluted with water, filtered, and passed through a mixed macroporous resin of type D101 and type AB-8. The resin was eluted with 50% ethanol as the eluent. The mixture was then concentrated under reduced pressure and spray-dried to obtain pomelo powder.

[0063] Test Example 1 This test example analyzes the components of the pomelo powder obtained in Example 1.

[0064] (1) The pomelo powder obtained in Example 1 was directly analyzed by HPLC. The chromatographic column was C18 (5 μm, 4.6×250 mm). The instrument was Alliance e2695.

[0065] Mobile phase: Phase A (acetonitrile), Phase B (0.1% FA); Column temperature: 30 ℃; Detection wavelength: 280 nm, 326 nm; Flow rate: 1.0 mL / min; Injection volume: 10 μL.

[0066] mobile phase gradient: HPLC chromatographic analysis results are as follows Figure 1 As shown, after comparison with the reference standard, the components in the pomelo powder were confirmed to be senna glycoside, neo-northern senna glycoside, rutin, naringin and neo-hesperidin.

[0067] (2) The pomelo powder obtained in Example 1 was separated by preparative high performance liquid chromatography, and the fraction was collected based on the main chromatographic peak with a retention time between 10 and 14 minutes.

[0068] Preparative high performance liquid chromatography purification: Instrument: High Performance Preparative Liquid Chromatography System 3 (HX-12789) Sample: HY-ZF-F10-P1 Preparation column: C18 (10 μm, 100 A, 100 DAC) Mobile phases: Phase A (acetonitrile), Phase B (water) Flow rate: 280 mL / min Mobile phase gradient: 10%A-80%A, 0-30 min The target fractions collected from multiple injections were combined, concentrated under reduced pressure at 40°C to remove most of the acetonitrile, and the remaining aqueous solution was freeze-dried to obtain five fractions with qualified purity.

[0069] The structures of the five pure products were identified, and their MS images are shown below. Figures 2-6 As shown, Figures 2-6 The mass spectra of senna-2, neo-North American senna-2, neo-hesperidin, naringin, and rutin are shown. The results indicate that all spectral data are consistent with the standard data reported in the literature, thus confirming that the five monomeric compounds are senna-2, neo-North American senna-2, neo-hesperidin, naringin, and rutin, respectively.

[0070] Test Example 2 This test case demonstrates the construction and validation of a high-throughput screening model for GLP-1 receptor agonists.

[0071] (1) The human GLP-1R gene sequence was found on NCBI, its sequence number was located, and corresponding primers were designed using Primer 5.0 software. The GLP-1R gene was synthesized using gene synthesis. It was then ligated into a vector by double enzyme digestion to construct the pLenti-GLP-1R-GFP-Puro overexpression vector. Logarithmically growing HEK-293T cells were collected, and a single-cell suspension was prepared using culture medium containing 10% fetal bovine serum. The cells were seeded into 6-well culture plates at a density of 2 × 10⁶ cells per well. 5 Cells were incubated per well at 37°C in a 5% CO2 incubator. On the second day, the original medium was replaced with 2 mL of fresh medium containing 5 μg / mL polybrene, and 20 μL of virus suspension was added. The cells were incubated at 37°C for 24 h, after which the medium in the wells was replaced with fresh medium. Cell fluorescence was observed 72 h after infection, and the results are as follows: Figure 7 As shown. After observing fluorescence, the culture medium in the 6-well plate was replaced with fresh medium containing 2 μg / mL puro. The selection medium was changed every 3-5 days according to the color of the medium and the cell growth.

[0072] (2) Take cells in the logarithmic growth phase, digest them with trypsin, and prepare a cell suspension with a density of 1000 cells / mL. Dilute it 10 times to 100 cells / mL, then dilute it 10 times again to 10 cells / mL. Seed 100 μL per well into a 96-well plate and incubate for 3-4 hours. After the cells adhere, observe the wells under a microscope, select wells with only one cell, mark them, and continue culturing. When the cell confluence reaches about 80-90%, digest them with trypsin and seed them sequentially into 24-well plates, 6-well plates, and 6 cm culture dishes. After screening, obtain monoclonal cell lines.

[0073] (3) Collect some cells, extract cellular proteins, and perform Western blotting to detect GLP-1R expression. The results are as follows: Figure 7 As shown in the WB results, the expression level of GLP-1R protein in the monoclonal line was significantly higher than that in the 293T cell group, indicating that the GLP-1R overexpressing monoclonal cell line was successfully constructed.

[0074] Test Example 3 This test example identifies the GLP-1 receptor agonist activity of pomelo powder obtained in Examples 1-2 and Comparative Examples 1-2.

[0075] Based on the drug screening cell model targeting GLP-1R constructed in Example 2, GLP-1R activation leads to an increase in cAMP levels. Intracellular cAMP concentration was directly and quantitatively measured using a commercially available cAMP detection kit. This method provides the most direct evidence of GLP-1R activation.

[0076] (1) The pomelo powder obtained in Examples 1-2 and Comparative Examples 1-2 was used as a sample to detect cytotoxicity.

[0077] HEK-293T cells and GLP-1R-HEK-293T cells were mixed at a ratio of 1×10⁻⁶. 4 Cells were seeded at a density of [number] cells per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours until complete cell adhesion. The old culture medium was discarded, and fresh culture medium containing the pomelo powder obtained in Examples 1-2 and Comparative Examples 1-2 was added to each well. A cell group without samples was set up as a blank control, and a cell-free culture medium group was set up as a zeroing well. After culturing for another 24 hours, 10 μL of CCK-8 solution was added to each well, and the plates were incubated for another 24 hours. The absorbance of each well was measured at 450 nm using a microplate reader.

[0078] like Figure 8 As shown in the figures, the first four graphs indicate that the concentration of pomelo powder in the range of 0-200 μg / mL has no significant effect on the survival rate of HEK-293T cells, and the latter four graphs indicate that the concentration of pomelo powder in the range of 0-200 μg / mL has no significant effect on the survival rate of GLP-1R-HEK-293T cells. Therefore, 100 μg / mL of pomelo powder was selected as the experimental concentration for subsequent experiments.

[0079] (2) Using the pomelo powder obtained in Examples 1-2 and Comparative Examples 1-2 as samples, the GLP-1 receptor agonist activity of each group of samples was detected.

[0080] HEK-293T cells and GLP-1R-HEK-293T cells were seeded in 96-well plates and cultured to an appropriate density. Then, the cells were starved using serum-free culture medium. Subsequently, blank control, 100 nm liraglutide, 100 μg / mL pomelo powder obtained in Examples 1-2, and Comparative Examples 1-2 were administered, respectively, and incubated for 4-6 hours. The culture medium was then discarded. A certain volume of cell lysate was taken and analyzed using a competitive ELISA method, following the specific operating procedures of the cAMP detection kit. Finally, the absorbance was read at a specific wavelength using a microplate reader, and the measured value was converted into intracellular cAMP concentration.

[0081] The test results are shown in Table 1. The results showed that after treatment with 100 μg / mL pomelo powder in Example 1, the cAMP concentration (pg / mL) in GLP-1R-HEK-293T cells was significantly higher than that in the blank control group, and there was no statistically significant difference compared to the Liraglutide group (P>0.05). The effect of pomelo powder in Example 2 was lower than that in Example 1. However, after changing the extraction method in Comparative Examples 1 and 2, the cAMP concentration was significantly lower than that in Example 1, indicating that the specific pomelo powder extraction method of this invention can obtain more effective active ingredients, and the resulting pomelo powder has the best effect in increasing cAMP levels. Test Example 4 This test case studies the intervention of pomelo powder obtained in Examples 1-2 and Comparative Examples 1-2 in obese mouse models.

[0082] I. Mouse weight monitoring Six-week-old ApoE- / - mice were randomly divided into a normal diet group (CTL), a high-fat diet modeling group (HFD), and a pre-modeling group for 6 weeks. A wild-type mouse group (WT) was also established and fed a normal diet throughout the treatment period.

[0083] The model groups were randomly divided into a model control group (HFD) and a drug treatment group. The drug treatment group was the pomelo powder group obtained in Examples 1-2 and Comparative Examples 1-2, respectively. Each group contained at least 8 mice, and they continued to be fed a high-fat diet for 8 weeks. The drug treatment group was given pomelo powder solution by gavage daily according to the weight of the mice (dose of 200 mg / kg / day), while the other three groups (CTL group, WT group, and HFD group) were given PBS solution.

[0084] During the 8-week treatment period, the mice's body weight was recorded weekly, and the experimental results were as follows: Figure 9As shown. The WT and CTL groups showed a steady increase in body weight; the HFD group mice were obese with abnormally high body weight; after 8 weeks of intervention, the mice in Example 1 showed a dose-dependent decrease in body weight, and the mice in Example 2 also showed a decrease in body weight, but the effect was worse than that in Example 1. The weight loss trend in Comparative Examples 1 and 2 was significantly slower, and their overall body weight was higher than that of Example 1 group.

[0085] II. Blood lipid level testing Mouse serum was collected and treated according to the instructions of the TC, TG, LDL-C, and HDL-C kits. The absorbance was measured at a specific wavelength using an ELISA reader, and the mouse blood lipid concentration was calculated.

[0086] The results are as follows Figure 10 As shown, compared to the WT group mice, the HFD group mice exhibited significant dyslipidemia, with elevated serum TC, TG, and LDL-C levels and decreased HDL-C levels. Compared to the HFD group mice, after treatment with pomelo powder in Example 1, serum TC decreased by 20%, TG decreased by 30.5%, LDL-C levels decreased by 44.5%, and HDL-C levels increased by 1.7 times. This indicates that pomelo powder can effectively improve HFD-induced dyslipidemia in mice and has certain potential to protect cardiovascular health. The effect of pomelo powder in Example 2 was lower than that in Example 1, and the effects of Comparative Examples 1-2 were significantly different from those in Example 1, indicating that pomelo powder obtained using the specific extraction method of this invention has a superior effect on improving dyslipidemia.

[0087] III. Blood Glucose Level Testing Mice were gently restrained, and blood was collected by pricking the tail tip. Fasting blood glucose was measured using a portable blood glucose meter and recorded as the 0-minute blood glucose value. Immediately afterward, mice were administered glucose solution by gavage at a dose of 2 g / kg body weight. Blood glucose levels were measured and recorded at 15, 30, 60, and 120 minutes after glucose administration. Prepared insulin solution was then injected intraperitoneally, typically at a volume of 5-10 mL / kg body weight. Blood glucose levels were measured and recorded at 15, 30, 60, 90, and 120 minutes after insulin injection.

[0088] The results are as follows Figure 11 As shown, in the Example 1 group of mice, the area under the glucose curve (AUC) at 120 minutes decreased by 15% in the glucose tolerance test and by 36.7% in the insulin tolerance test. This indicates that grapefruit powder treatment can effectively improve glucose metabolism disorders and enhance insulin sensitivity in mice. The effects of Example 2 and Comparative Examples 1-2 were lower than those of Example 1, but also showed a decreasing trend.

[0089] IV. Histopathological staining of liver sections In the WT group, CTL group, HFD group, and Example 1 group, mouse livers were randomly selected for H&E staining (HE). Figure 12 As shown, compared with the WT group mice, the HFD group mice exhibited numerous round vacuoles of varying sizes in their liver tissue structure, and some hepatocytes were significantly enlarged and rounded. In contrast, compared with the HFD group, the aforementioned pathological changes in the liver tissue of the mice in Example 1 group were significantly improved, with clearer liver lobule structures and a marked reduction in fat vacuole volume.

[0090] V. Pathological staining of white adipose tissue In the WT group, CTL group, HFD group, and Example 1 group, white adipose tissue from mice was randomly selected for Oil Red O staining (ORO). Figure 12 As shown, compared with the WT group mice, the HFD group mice had larger white adipocytes, while compared with the HFD group, the adipocytes in the Example 1 group decreased in size and lipid staining became lighter after treatment.

[0091] VI. Histopathological staining of aortic root sections In the WT group, CTL group, HFD group, and Example 1 group, mouse aortic samples were randomly selected from each group, fixed, and then heart samples were fixed, dehydrated, embedded, and frozen sections of the aortic root were prepared, followed by HE staining and ORO staining. Figure 12 As shown, HE staining and ORO staining results of aortic root sections showed that pomelo powder significantly reversed HFD-induced plaque area. The plaque area in the Example 1 group was reduced by 40% compared with the model group, and the collagen content in the plaque was increased, and the stability was enhanced.

[0092] VII. Research on the mechanism by which pomelo powder improves cardiovascular disease by activating GLP-1 receptors Grapefruit powder, as a natural GLP-1R agonist, primarily acts in the gut; by activating GLP-1R in intestinal L cells and local intestinal tissues, it initiates the cAMP / PKA / CREB signaling pathway, thereby improving systemic metabolism. The mechanism of action of the grapefruit powder obtained in Example 1 was investigated.

[0093] Total protein was extracted from colon tissue and quantified using the BCA method. 10 μg of protein was loaded into each well, and after SDS-PAGE electrophoresis, the sample was transferred to a membrane. The membrane was incubated overnight with primary antibodies against p-CREB, total CREB, anti-GLP-1R, and the internal control anti-β-Actin. The following day, the membrane was incubated with secondary antibodies and then subjected to ECL chemiluminescence imaging.

[0094] The results are as follows Figure 13As shown, compared with the HFD group, the GLP-1R protein expression level of the pomelo powder group obtained in Example 1 was significantly increased, and the CREB phosphorylation level (p-CREB / total CREB) was significantly upregulated, confirming that pomelo powder exerts a metabolic improvement effect by activating the GLP-1R / cAMP / PKA / CREB signaling pathway.

[0095] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0096] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0097] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. The application of a grapefruit powder or juice targeting the GLP-1 receptor in the preparation of products for weight loss and improvement of blood sugar and lipid disorders, characterized in that, The pomelo powder or pomelo juice is obtained by a preparation method including the following steps: After ultrasonic-assisted enzymatic hydrolysis, the raw material of pomelo is extracted by reflux. After extraction, the filtrate is concentrated and purified to obtain pomelo powder or pomelo juice.

2. The application according to claim 1, characterized in that, The raw materials for pomelo include any one or a combination of at least two of the following: pomelo peel, pomelo pulp, and pomelo seeds; Preferably, the pomelo raw material is selected from a combination of pomelo peel and pomelo pulp; Preferably, the solvent used for the ultrasound-assisted enzymatic hydrolysis is an ethanol-water solution with a volume fraction of 30-50%; Preferably, during the ultrasound-assisted enzymatic hydrolysis, the ratio of pomelo raw material to solvent is 1:(8-20) g / mL; Preferably, the ultrasonic power of the ultrasound-assisted enzymatic hydrolysis is 200-400 W; Preferably, the ultrasound-assisted enzymatic hydrolysis is performed using a complex enzyme of cellulase, pectinase, and β-glucosidase in a mass ratio of (0.5-3):(0.5-2):

1. Preferably, the compound enzyme accounts for 3-6% of the mass of the pomelo raw material; Preferably, the temperature for ultrasound-assisted enzymatic hydrolysis is 40-55℃ and the time is 30-60 min.

3. The application according to claim 1 or 2, characterized in that, The reflux extraction conditions are as follows: anhydrous ethanol or an aqueous ethanol solution is added to the system until the final concentration of ethanol is 55-70%. Preferably, the reflux extraction time is 1-3 hours; Preferably, the separation and purification method includes macroporous adsorption resin column chromatography; Preferably, the macroporous adsorption resin includes any one or a combination of at least two of the following types: D101, AB-8, HPD-100, and XAD-7. Preferably, the macroporous adsorption resin is selected from a combination of D101 and AB-8 types.

4. Application of pomelo powder or pomelo juice in the preparation of GLP-1 receptor agonists.

5. The application according to claim 4, characterized in that, The pomelo powder or pomelo juice is obtained by a preparation method including the following steps: After ultrasonic-assisted enzymatic hydrolysis, the raw material of pomelo is extracted by reflux. After extraction, the filtrate is concentrated and purified to obtain pomelo powder or pomelo juice.

6. The application according to claim 4 or 5, characterized in that, The solvent used in the ultrasound-assisted enzymatic hydrolysis is an ethanol-water solution with a volume fraction of 30-50%. Preferably, the ultrasound-assisted enzymatic hydrolysis is performed using a complex enzyme of cellulase, pectinase, and β-glucosidase in a mass ratio of (0.5-3):(0.5-2):

1. Preferably, the temperature for the ultrasound-assisted enzymatic hydrolysis is 40-55℃, and the time is 30-60 min; The reflux extraction conditions are as follows: anhydrous ethanol or an aqueous ethanol solution is added to the system until the final concentration of ethanol is 55-70%. Preferably, the separation and purification method includes macroporous adsorption resin column chromatography; Preferably, the macroporous adsorption resin includes any one or a combination of at least two of the following types: D101, AB-8, HPD-100, and XAD-7. Preferably, the macroporous adsorption resin is selected from a combination of D101 and AB-8 types.

7. The use of pomelo powder or pomelo juice in the preparation of products for the prevention or treatment of metabolic diseases, characterized in that, The pomelo powder or pomelo juice is obtained by a preparation method including the following steps: After ultrasonic-assisted enzymatic hydrolysis, the raw material of pomelo is extracted by reflux. After extraction, the filtrate is concentrated and purified to obtain pomelo powder or pomelo juice.

8. The application according to claim 7, characterized in that, The metabolic diseases mentioned include any one of obesity, atherosclerosis, hyperlipidemia, non-alcoholic fatty liver disease, and metabolic syndrome.

9. The application according to claim 7 or 8, characterized in that, The solvent used in the ultrasound-assisted enzymatic hydrolysis is an ethanol-water solution with a volume fraction of 30-50%. Preferably, the ultrasound-assisted enzymatic hydrolysis is performed using a complex enzyme of cellulase, pectinase, and β-glucosidase in a mass ratio of (0.5-3):(0.5-2):

1. Preferably, the temperature for the ultrasound-assisted enzymatic hydrolysis is 40-55℃, and the time is 30-60 min; The reflux extraction conditions are as follows: anhydrous ethanol or an aqueous ethanol solution is added to the system until the final concentration of ethanol is 55-70%. Preferably, the separation and purification method includes macroporous adsorption resin column chromatography; Preferably, the macroporous adsorption resin includes any one or a combination of at least two of the following types: D101, AB-8, HPD-100, and XAD-7. Preferably, the macroporous adsorption resin is selected from a combination of D101 and AB-8 types.

10. The application according to any one of claims 7-9, characterized in that, The products used for the prevention or treatment of metabolic diseases also include pharmaceutically acceptable excipients. Preferably, the excipients include any one or a combination of at least two of the following: carrier, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer. Preferably, the pomelo powder or pomelo juice constitutes 5-50% by mass in the product for the prevention or treatment of metabolic diseases.

Images