Corn extract promoting secretion of glp-1 and its preparation method and application

Abstract

The application belongs to the field of functional substance research, and particularly relates to a corn extract for promoting GLP-1 secretion, and a preparation method and application thereof. The corn extract provided by the application is a dried product obtained by ultrasonic extraction of a dried powder of fresh corn kernels after room temperature soaking in ethanol or an ethanol aqueous solution; the fresh corn comprises yellow sweet corn; the temperature of the ultrasonic extraction is 30-60 DEG C; the power of the ultrasonic extraction is 50-150 W; and the time of the ultrasonic extraction is 30-90 min. The preparation method of the corn extract is simple and easy to implement, and can effectively reduce the production cost of functional ingredients. The corn extract can effectively promote the secretion of GLP-1 by intestinal L cells in both healthy and insulin-resistant states, and simultaneously improve cell viability.

Description

Technical Field

[0001] This invention belongs to the field of functional substance research, specifically relating to corn extract that promotes GLP-1 secretion, its preparation method, and its application. Background Technology

[0002] In recent years, the global prevalence of diabetes has continued to rise, reaching 8.8% of the total population in adults worldwide in 2017, and is projected to further increase to 9.9% by 2045. Glucagon-like peptide-1 (GLP-1) is a multifunctional hormone with various physiological functions, mainly secreted by L cells in the distal intestine. After its release, it plays a key role in promoting insulin secretion, regulating blood glucose homeostasis, and controlling appetite. GLP-1 is produced by enzymatic cleavage of proglucagon protein. Its precursor gene is derived from the proglucagonogen gene (GCG), and its cleavage enzyme is proprotein convertase-1 (PCSK-1).

[0003] L cells are open-cell endocrine cells that can be directly stimulated by nutrients in the intestinal lumen. Mouse and human L cell lines (STC-1, GLUTag, and NCI-H716) are widely used as cell models to study the effects of dietary components on intestinal hormone secretion. For example, Reference 1 discloses the preparation and application of a whey protein-derived hypoglycemic peptide with GLP-1 receptor agonist activity. This peptide, prepared from bovine whey protein, is gastrointestinally digestible and absorbable, and can promote GLP-1 secretion in NCI-H716 cells. Reference 2 discloses a *Huangda* oligosaccharide with GLP-1 agonist activity and its application; the *Huangda* oligosaccharide fragment obtained through glycosidase degradation can increase the level of GLP-1 secreted by STC-1 cells. Reference 3 discloses the application of berberine metabolites in the preparation of drugs that stimulate GLP-1 secretion, finding that berberine metabolites (including berberine and / or palmatine) can promote GLP-1 secretion in GLUTag cells. Reference 4 tested the GLP-1 stimulation effect of 21 novel O-diarylheptane dimers (kaempferiae A1-A4, B1-B13 and C1-C4) in the ethyl acetate fraction of Kaempferiae Rhizoma on NCI-H716 cells.

[0004] While using L-cell assays to assess the effects of dietary components on intestinal hormone secretion is a common experimental method, most current experimental systems directly employ healthy intestinal cell models to evaluate the direct stimulatory effects of active ingredients on intestinal hormone secretion. Although such studies can elucidate basic secretion mechanisms, they fail to simulate the pathological microenvironment of insulin resistance under specific physiological conditions such as diabetes. Because insulin resistance significantly alters cellular responses to nutrient signals and hormonal regulation, conclusions drawn solely from healthy cell models cannot fully reflect the actual efficacy and molecular mechanisms of active ingredients in insulin resistance, a core physiological aspect of diabetes.

[0005] The role of food in lowering blood glucose levels and slowing the progression of type 2 diabetes mellitus (T2DM) is receiving increasing attention, with its mechanism of action likely involving the regulation of gastrointestinal hormones. Reference 5 discloses that ellagitannins extracted from blackcurrant seeds alleviated dextran sodium (DSS)-induced colonic inflammation in mice and significantly increased total intestinal GLP-1 secretion and GLP-1 receptor mRNA expression levels. Reference 6 discloses that a mixture of polyphenols and other compounds isolated from apple peel and pulp residue can promote GLP-1 transcription and secretion, improving glucose tolerance. Reference 7 discloses six novel alkaloids (compounds 1-6) isolated from purslane; treatment of STC-1 cells with these alkaloids induced GLP-1 secretion, suggesting that these alkaloids may lower blood glucose levels by stimulating GLP-1 release.

[0006] Whole grains, rich in dietary fiber, resistant starch, and phenolic compounds, play a crucial role in the prevention of type 2 diabetes mellitus (T2DM). Previous studies have confirmed a negative correlation between T2DM incidence and whole grain intake. Therefore, the regulatory effect of whole grains and their derivatives on GLP-1 has become a research hotspot. Reference 8 discloses an oat-derived active polypeptide with GLP-1-promoting, hypoglycemic, and lipid-lowering activities, along with its preparation method and applications. This method uses oat bran as a raw material to prepare an oat polypeptide with GLP-1-promoting, hypoglycemic, and lipid-lowering activities. Reference 9 discloses an oat-derived GLP-1-promoting oligopeptide, its preparation method, and its applications. This oligopeptide can be artificially synthesized through genetic engineering, directly obtained from oat protein through separation and purification, or directly prepared through chemical synthesis. Reference 10 discloses a composition for assisting in lowering blood sugar and improving insulin resistance, comprising depolymerized complex cereal fibers, including wheat fiber, corn fiber, sweet potato fiber, and pea fiber.

[0007] Existing research largely focuses on the effects of whole grains and their individual components on GLP-1 secretion, neglecting the synergistic effects among various active ingredients. Furthermore, most studies emphasize the role of whole grain bioactive factors in regulating blood glucose, while research on the GLP-1 secretion-promoting mechanism is relatively limited.

[0008] Corn, as the most widely cultivated cereal crop globally, possesses significant nutritional value and health benefits. Sweet corn, in particular, stands out as a top-tier variety of corn, frequently used as a raw material for processed foods due to its high nutritional value. Currently, research has been conducted on different types of corn and their derivatives. For example, reference 11 discloses an extraction method for purple corn kernels, utilizing ethanol extraction under ultrasonic conditions. The resulting extract can inhibit renal fibrosis and glomerulosclerosis caused by chronic hyperglycemia. Reference 12 discloses a multigrain composition beneficial for weight loss and its preparation method. The composition includes quinoa, Job's tears, oats, millet, and black glutinous corn extracts, wherein the black glutinous corn extract is obtained by ethanol extraction and ethyl acetate extraction of black glutinous corn kernels. Reference 13 discloses a plant extract for lowering blood lipids and blood pressure and its preparation process. The plant extracts include perilla extract, apocynum extract, ginkgo leaf extract, corn extract, coptis extract, and cattail pollen extract, wherein the corn extract is a corn gluten meal extract. In research on the anti-diabetic effects of corn, studies have shown that selenium polysaccharides from sweet corn cobs effectively lower blood sugar levels and reduce insulin resistance by decreasing intestinal barrier permeability, reducing inflammatory factors, and regulating gut microbiota. Purple corn extract, rich in anthocyanins, exerts its anti-diabetic effect by protecting pancreatic β-cells and promoting insulin secretion.

[0009] Although there have been studies on corn, no research has yet clarified whether corn is effective in promoting GLP-1 secretion, and its secretion mechanism has not been reported.

[0010] References:

[0011] Reference 1: CN120699089A.

[0012] Reference 2: CN120025387B.

[0013] Reference 3: CN116139134B.

[0014] Reference 4: Antidiabetic constituents of Kaempferiae rhizoma: Previously undescribed O-linked diarylheptanoid dimers promoting GLP-1 secretion via PKA-CREB pathway.

[0015] Reference 5: Anti-inflammatory activities of black raspberry seedellagitannins and their structural effects on the stimulation of glucagon-like peptide-1 secretion and intestinal bitter taste receptors.

[0016] Reference 6: Apple Polyphenols Extracts Ameliorate High CarbohydrateDiet-Induced Body Weight Gain by Regulating the Gut Microbiota and Appetite.

[0017] Reference 7: Identification of Novel Alkaloids from Portulaca oleraceaL. and Characterization of Their Pharmacokinetics and GLP-1 Secretion-Promoting Activity in STC-1 Cells.

[0018] Reference 8: CN119736363B.

[0019] Reference 9: CN115353551B.

[0020] Reference 10: CN121003667A.

[0021] Reference 11: KR101063524B1.

[0022] Reference 12: CN111513249A.

[0023] Reference 13: CN118001345A. Summary of the Invention

[0024] The problem the invention aims to solve

[0025] While existing technologies (such as those cited in references 1-13 above) have studied foods and their derivatives that are beneficial to blood sugar health, such research is still insufficient. Most studies focus on the extraction and identification of single, high-purity components. These extraction processes are not only cumbersome and energy-intensive, but also result in high final product costs, making them unsuitable for the preparation of functional foods or pharmaceuticals. Furthermore, most existing research is based on healthy L cells and generally lacks studies on insulin resistance, a crucial physiological state, thus failing to truly reflect the potential efficacy of foods and their derivatives under specific physiological conditions such as diabetes. At the same time, the types of foods and their derivatives covered by existing technologies remain limited, especially whole grains and their derivatives. For example, while there are studies on whole grains such as corn kernels, most focus on dark corn rich in anthocyanins.

[0026] This invention has conducted extensive research to provide an extract with significant GLP-1 secretion-promoting activity isolated from naturally occurring and readily available yellow sweet corn kernels, along with its preparation method and applications. This extract effectively promotes GLP-1 secretion by L cells in insulin-resistant states, and its preparation method is simple. It addresses the problems of existing methods, such as the lack of corn coverage, complex extraction processes, and high costs. This provides a solid material basis for developing safer sources of nutritional intervention strategies that help maintain healthy blood glucose levels, particularly suitable for diabetic populations, and offers theoretical support for the research and development of whole-grain foods.

[0027] Solution for solving the problem

[0028] [1]. A corn extract that helps promote GLP-1 secretion, wherein the corn extract is a dried product obtained by ultrasonic extraction of dried powder of fresh corn kernels after soaking in ethanol or ethanol aqueous solution at room temperature; the fresh corn includes yellow sweet corn; the ultrasonic extraction temperature is 30~60℃; the ultrasonic extraction power is 50~150W; and the ultrasonic extraction time is 30~90min.

[0029] [2]. According to the corn extract described in [1], the dried powder of the fresh corn kernels is prepared by the following steps: take fresh corn kernels, wash them, freeze-dry them under vacuum, pulverize them and pass them through a 40-80 mesh sieve, and place the sieved powder in a desiccator to balance it, and that is the result.

[0030] [3]. The corn extract according to [1] or [2], wherein the concentration of the aqueous ethanol solution is ≥70% (v / v).

[0031] [4]. The corn extract according to any one of [1] to [3], wherein the soaking time at room temperature is 15 to 30 min.

[0032] [5]. The corn extract according to any one of [1] to [4], wherein the variety of the yellow sweet corn includes at least one of Overland, Saint Sweet 1365 and SBS903.

[0033] [6]. The method for preparing corn extract according to any one of [1] to [5], wherein the method comprises the following steps: taking fresh corn kernels, making them into dried powder, weighing the powder and mixing it with ethanol or an aqueous ethanol solution at a mass-volume ratio of (0.5-5):10, soaking it at room temperature and then performing ultrasonic treatment, separating the solid and liquid to obtain a supernatant and a precipitate, repeating the operation of mixing with ethanol or an aqueous ethanol solution at a mass-volume ratio of (0.5-5):10, soaking at room temperature, ultrasonic treatment and solid-liquid separation on the precipitate once, twice or more, combining all the obtained supernatants and performing vacuum concentration and freeze drying to obtain the extract.

[0034] [7]. According to the method described in [6], the vacuum concentration is carried out by means of a rotary evaporator under water bath conditions of 35~45°C.

[0035] [8]. Use of corn extract according to any one of [1] to [5] in the preparation of products that help maintain healthy blood glucose levels and / or help promote the secretion of GLP-1 by intestinal L cells.

[0036] [9]. According to the use described in [8], wherein the aid in maintaining healthy blood glucose levels includes aiding in maintaining healthy blood glucose levels in a state of insulin resistance, and / or, the aid in promoting the secretion of GLP-1 by intestinal L cells includes aiding in promoting the secretion of GLP-1 by intestinal L cells in a state of insulin resistance.

[0037]

[10] . According to the use described in [8] or [9], wherein the ability to maintain healthy blood glucose levels and / or to promote the secretion of GLP-1 by intestinal L cells includes any one or more of the following: increasing cAMP levels in intestinal L cells, increasing calcium ion concentration in intestinal L cells, increasing Gcg gene expression in intestinal L cells, increasing Pcsk1 gene expression in intestinal L cells, increasing phosphorylation level of PKA protein in intestinal L cells, increasing phosphorylation level of CaMKII protein in intestinal L cells, increasing phosphorylation level of ERK protein in intestinal L cells, and increasing phosphorylation level of CREB protein in intestinal L cells.

[0038]

[11] . Use of corn extract according to any one of [1] to [5] in the preparation of products having any one or more of the following effects: increasing cAMP content in intestinal L cells, increasing calcium ion concentration in intestinal L cells, increasing Gcg gene expression in intestinal L cells, increasing Pcsk1 gene expression in intestinal L cells, increasing phosphorylation level of PKA protein in intestinal L cells, increasing phosphorylation level of CaMKII protein in intestinal L cells, increasing phosphorylation level of ERK protein in intestinal L cells, and increasing phosphorylation level of CREB protein in intestinal L cells.

[0039]

[12] . Use of corn extract according to any one of [1] to [5] in the preparation of products that help improve the reduction of GLP-1 secretion by intestinal L cells due to insulin resistance.

[0040]

[13] . Use of corn extract according to any one of [1] to [5] in the preparation of products that help prevent and / or improve insulin resistance.

[0041]

[14] . The use according to any one of [8] to

[13] is characterized in that the product includes food.

[0042] The effects of the invention

[0043] This invention provides an extract with significant GLP-1 secretion-promoting activity isolated from naturally available sweet yellow corn kernels, along with its preparation method and applications. First, the preparation method of the corn extract provided by this invention is simple and easy to implement, effectively reducing the production cost of functional components. Second, the corn extract prepared using the method provided by this invention not only enhances the ability of intestinal L cells to secrete GLP-1 under healthy conditions, but also effectively promotes GLP-1 secretion by intestinal L cells under insulin resistance, while simultaneously increasing cell viability. Experimental data show that its mechanism of action involves increased intracellular cAMP and cytoplasmic calcium ion levels, and upregulation of GLP-1-related genes (Gcg and Pcsk1). Simultaneously, the phosphorylation levels of key signaling proteins related to GLP-1 secretion (including PKA, CaMKII, ERK, and CREB) are significantly increased due to the intervention of this corn extract, demonstrating that the corn extract prepared by the method provided by this invention can effectively activate the GLP-1 secretion pathway in intestinal L cells. This provides a solid material basis for developing products with safer sources that help maintain healthy blood sugar levels, especially suitable for diabetics, and also provides theoretical support for the research and development of whole grain foods. Attached Figure Description

[0044] Figure 1Effects of different concentrations of palmitic acid on the viability of STC-1 cells.

[0045] Figure 2 Effects of different concentrations of corn extract on the viability of STC-1 cells.

[0046] Figure 3 Effects of different concentrations of corn extract on STC-1 cell viability under palmitic acid intervention.

[0047] Figure 4 Effects of different concentrations of corn extract on GLP-1 secretion in STC-1 cells under normal conditions.

[0048] Figure 5 Effects of extracts from different corn varieties on GLP-1 secretion in STC-1 cells under normal conditions.

[0049] Figure 6 Effects of different concentrations of maize extract on GLP-1 secretion in STC-1 cells under palmitic acid intervention.

[0050] Figure 7 Effects of different concentrations of maize extract on cAMP levels in STC-1 cells under palmitic acid intervention.

[0051] Figure 8 Effects of different concentrations of corn extract on cytoplasmic calcium in STC-1 cells under palmitic acid intervention.

[0052] Figure 9 Effects of different concentrations of maize extract under palmitic acid intervention on the expression of Gcg and Pcsk1 genes in STC-1 cells.

[0053] Figure 10 Effects of different concentrations of maize extract under palmitic acid intervention on the expression of GLP-1 secretion-related proteins in STC-1 cells. Detailed Implementation

[0054] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0055] In this invention, the terms "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to the described embodiment that are included in at least one embodiment described herein and may or may not exist in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0056] In this invention, the numerical range expressed as "value A ~ value B" or "value A - value B" refers to the range that includes the endpoints values ​​A and B. In this invention, the numerical range expressed as "above value A" and "below value A" includes value A.

[0057] In this invention, the word "may" has both the meaning of performing a certain process and the meaning of not performing a certain process. In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the case where the event occurs and the case where the event does not occur.

[0058] In this invention, the term "about" can mean that a value includes the standard deviation of the error of the apparatus or method used to determine that value. The numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains a standard deviation due to the aforementioned testing apparatus or method. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified with "about". Here, "about" generally means that the actual value is within ±5%, ±1%, or ±0.5% of a particular value or range.

[0059] In this invention, the term "comprising" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that comprises a series of steps or units is not limited to the steps or units listed, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such processes, methods, products, or apparatus.

[0060] In this invention, the term "room temperature" refers to 23±2℃.

[0061] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0062] This invention is primarily based on the following concept: It provides a method for preparing a yellow sweet corn extract. Based on the prepared corn extract, its effect on GLP-1 secretion by intestinal L cells (such as STC-1 cells) under normal conditions was investigated. The survival rate, GLP-1 secretion level, cAMP level, cytoplasmic calcium ion level, and gene and protein expression levels of the extract in palmitic acid-induced intestinal L cells (such as STC-1 cells) were also examined. Experimental data show that the corn extract prepared by this invention can effectively improve the survival rate and GLP-1 secretion level of intestinal L cells (such as STC-1 cells). The increased GLP-1 secretion may be achieved by upregulating cAMP levels, cytoplasmic calcium levels, and the levels of GLP-1 secretion-related genes and proteins. In short, this invention provides a method for preparing a yellow sweet corn extract. Experiments have shown that this yellow sweet corn extract has anti-diabetic activity. Furthermore, this invention explores its mechanism of action in promoting GLP-1 secretion, providing new ideas for using corn to prepare products that help maintain healthy blood sugar levels and improve or prevent insulin resistance. The invention will be described in more detail below.

[0063] I. Preparation method of corn extract

[0064] This invention provides a method for preparing a corn extract that helps promote GLP-1 secretion. The method includes the following steps: taking fresh corn kernels, making them into a dry powder, weighing the powder and mixing it with ethanol or an aqueous ethanol solution at a mass-volume ratio of (0.5-5):10, soaking it at room temperature, and then performing ultrasonic treatment. After solid-liquid separation, a supernatant and a precipitate are obtained. The operation of mixing with ethanol or an aqueous ethanol solution at a mass-volume ratio of (0.5-5):10, soaking at room temperature, ultrasonic treatment, and solid-liquid separation is repeated once, twice, or more (preferably twice) on the precipitate. All the obtained supernatants are combined and concentrated under reduced pressure and freeze-dried to obtain the final product.

[0065] In some embodiments, the fresh corn includes, preferably, yellow sweet corn. Yellow sweet corn is widely cultivated, readily available, and inexpensive to obtain.

[0066] In some embodiments, the yellow sweet corn varieties selected in this invention include at least one of Overland, Saint Sweet 1365 and SBS903; preferably, at least one of Overland and SBS903; more preferably, Overland or SBS903.

[0067] In some embodiments, the dried powder is prepared by the following steps: taking fresh corn kernels, washing them, freeze-drying them under vacuum, pulverizing them, and passing them through a 40-80 mesh sieve, preferably a 60 mesh sieve. The powder passing through the sieve is then placed in a desiccator for equilibration, and the powder is obtained.

[0068] In some specific implementations, the freeze-drying time is 24-72 hours, preferably 48 hours, to completely remove moisture from the corn kernels using the basic method, thereby obtaining a loosely textured freeze-dried corn product.

[0069] In some specific implementation schemes, the pulverization can be initially carried out using a high-speed pulverizer, followed by further processing with a ball mill to make the powder fine and uniform.

[0070] In some specific implementations, the equilibration time in the dryer is 30 minutes.

[0071] Freeze-drying removes moisture, making the corn kernels loose and porous, greatly increasing the efficiency of subsequent grinding. Proper grinding and sieving increase the specific surface area, and uniformly sized powder facilitates solvent penetration, improving the extraction efficiency of functional components. Dryer balancing ensures consistent powder moisture content, preventing weighing errors or moisture interference with extraction efficiency caused by varying environmental humidity.

[0072] In some embodiments, the concentration of the aqueous ethanol solution is ≥70% (v / v).

[0073] In some preferred embodiments, 100% ethanol is used to mix with the powder.

[0074] In some embodiments, the mass-volume ratio of the powder or precipitate to ethanol or an aqueous ethanol solution is (0.5-4):10, preferably (0.5-3):10, more preferably (0.5-2.5):10, and even more preferably (0.5-1.5):10.

[0075] In some embodiments, the room temperature soaking time is 15-30 min, preferably 20 min, and preferably with slight oscillation during soaking; the ultrasonic treatment temperature is 30-60°C, preferably 30-50°C, more preferably 40°C; the ultrasonic treatment power is 50-150W, preferably 75-125W, more preferably 87-113W; the ultrasonic treatment time is 30-90 min, preferably 40-80 min, more preferably 50-70 min; the ultrasonic pulse is 5-15 s / 2 s pause, preferably 10 s / 2 s pause.

[0076] Soaking and agitation allow the powder to be fully wetted and penetrated, preventing clumping and preparing it for subsequent efficient ultrasonic extraction. Proper ultrasonic treatment utilizes the cavitation effect of ultrasound to generate localized high temperature and pressure, breaking down cell walls and accelerating the release of functional components from the cells into the extraction solvent, significantly shortening extraction time and improving extraction efficiency.

[0077] Repeated extraction can maximize the extraction of functional components remaining in the precipitate, thereby increasing the overall yield.

[0078] In some embodiments, the solid-liquid separation is performed by centrifugation, preferably at 10,000 rpm for 10 min.

[0079] In some embodiments, the supernatant is further filtered using a microporous membrane before being concentrated under reduced pressure, preferably with a pore size of 0.45 μm.

[0080] Filtration removes fine particles, suspended impurities, or large colloids from the supernatant, resulting in a clear filtrate that protects the rotary evaporator.

[0081] In some embodiments, the vacuum concentration is carried out using a rotary evaporator in a water bath at 35-45°C, preferably at 40°C, until most of the ethanol is recovered and the supernatant is in a concentrated state.

[0082] By using a rotary evaporator for low-temperature, reduced-pressure concentration, not only can ethanol be recovered and reused, but the volume of the extract can also be reduced, increasing the concentration of functional substances and facilitating subsequent freeze-drying. Reduced pressure lowers the boiling point, preventing component decomposition caused by prolonged high-temperature heating.

[0083] In some embodiments, after the supernatant is concentrated under reduced pressure, it is first frozen at -80°C until completely solidified, and then placed in a freeze dryer for vacuum freeze drying for 14-72 hours, preferably 48 hours.

[0084] By freeze-drying the supernatant after vacuum concentration, residual trace amounts of moisture and extraction solvent can be removed, yielding a completely dry solid powder. Freeze-drying also preserves the activity of the components, and the low-temperature process avoids decomposition, oxidation, or deterioration that may occur with heat drying. The final product is porous and easily dissolved, weighed, stored, and transported.

[0085] The method for preparing corn extract provided by this invention is simple and easy to implement, using uncomplicated raw materials, which greatly reduces the extraction cost of functional components from corn. Furthermore, the entire preparation process of corn extract provided by this invention is carried out in a relatively low-temperature environment, avoiding the loss of heat-sensitive components and enabling efficient and complete separation of alcohol-soluble active ingredients from corn.

[0086] II. Corn extract

[0087] This invention provides a corn extract that promotes GLP-1 secretion through the above-described preparation method. Specifically, the corn extract provided by this invention is a dried product obtained by ultrasonic extraction of dried powdered fresh corn kernels after soaking in ethanol or an ethanol-water solution at room temperature. The fresh corn includes yellow sweet corn; preferably, the yellow sweet corn variety includes at least one of Auveran, Saint Sweet 1365, and SBS903; more preferably, the yellow sweet corn variety includes at least one of Auveran and SBS903; even more preferably, the yellow sweet corn variety is Auveran, Saint Sweet 1365, or SBS903, or the yellow sweet corn variety is Auveran or SBS903.

[0088] Compared to the single active ingredients (such as polyphenols or fatty acids with specific structures) that help promote GLP-1 secretion in the prior art, the corn extract provided by the present invention is a complex with multiple functional components, synergistic effects between the components, and comprehensive effects, which breaks through the limitations of the prior art in terms of raw material selection and component composition.

[0089] III. Uses of Corn Extract

[0090] This invention provides the use of the above-described corn extract in the preparation of products that help maintain healthy blood glucose levels and / or help promote the secretion of GLP-1 by intestinal L cells. In some embodiments, the "helping maintain healthy blood glucose levels" includes helping to maintain healthy blood glucose levels in a state of insulin resistance, and / or, the "helping promote the secretion of GLP-1 by intestinal L cells" includes helping to promote the secretion of GLP-1 by intestinal L cells in a state of insulin resistance. In some embodiments, this invention provides the use of the above-described corn extract in the preparation of products that help improve the reduction of GLP-1 secretion by intestinal L cells caused by insulin resistance.

[0091] In some embodiments, the methods described in this invention for helping to maintain healthy blood glucose levels, helping to maintain healthy blood glucose levels in a state of insulin resistance, helping to promote the secretion of GLP-1 by intestinal L cells, helping to promote the secretion of GLP-1 by intestinal L cells in a state of insulin resistance, and improving the reduction of GLP-1 secretion by intestinal L cells caused by insulin resistance are not intended to prevent and / or treat diseases.

[0092] The present invention also provides the use of the above-mentioned corn extract in the preparation of products that help prevent and / or improve insulin resistance.

[0093] In some implementations, the benefits of maintaining healthy blood glucose levels and / or promoting GLP-1 secretion from intestinal L cells and / or improving insulin resistance-induced reduction in GLP-1 secretion from intestinal L cells include any one or more of the following: increasing cAMP levels in intestinal L cells, increasing calcium ion concentration in intestinal L cells, increasing Gcg gene expression in intestinal L cells, increasing Pcsk1 gene expression in intestinal L cells, increasing phosphorylation levels of PKA protein in intestinal L cells, increasing phosphorylation levels of CaMKII protein in intestinal L cells, increasing phosphorylation levels of ERK protein in intestinal L cells, and increasing phosphorylation levels of CREB protein in intestinal L cells.

[0094] In some implementations, the benefits of maintaining healthy blood glucose levels and / or promoting GLP-1 secretion from intestinal L cells and / or improving insulin resistance-induced reduction in GLP-1 secretion from intestinal L cells include increasing cAMP levels in intestinal L cells, increasing calcium ion concentration in intestinal L cells, increasing Gcg gene expression in intestinal L cells, increasing Pcsk1 gene expression in intestinal L cells, increasing phosphorylation levels of PKA protein in intestinal L cells, increasing phosphorylation levels of CaMKII protein in intestinal L cells, increasing phosphorylation levels of ERK protein in intestinal L cells, and increasing phosphorylation levels of CREB protein in intestinal L cells.

[0095] In some embodiments, the corn extract in the product, on a dry weight basis, is greater than 0.001%; ​​preferably greater than 0.01%.

[0096] In some implementations, the product includes food.

[0097] In some implementations, the food is in the form of a liquid or a solid.

[0098] In some embodiments, the food includes confectionery (including, for example, hard candy, gel candy, shortbread candy, compressed candy, and aerated candy), beverages (including, for example, carbonated beverages, tea beverages, coffee beverages, fruit and vegetable juice beverages, and lactic acid bacteria beverages), dairy products (including, for example, milk powder, cheese, yogurt, and liquid milk), and / or baked goods (including, for example, hard capsules, soft capsules, tablets, oral liquids, pills, granules, and powders).

[0099] In some implementations, the food is a health food, which can be a hard capsule, soft capsule, tablet, oral liquid, pill, granule or powder.

[0100] In some embodiments, the corn extract is present in a content greater than 0.01 wt% in the food.

[0101] In addition to the corn extract mentioned above, depending on the type of product and the end needs of the target audience, in some embodiments, the food may also contain one or more of the following ingredients: plant-based ingredients, animal dairy ingredients, animal meat ingredients, functional additives, and any acceptable excipients.

[0102] Plant-based ingredients can include, for example, fruits such as fig, pomegranate, kiwi, orange, tangerine, pineapple, strawberry, apple, banana, grape, pear, cherry, blueberry, blackberry, blackcurrant, cranberry, raspberry, melon, amla, and bilberry, or their extracts; fruits and vegetables such as onion, cucumber, tomato, cauliflower, carrot, spinach, kale, Brussels sprouts, garlic, basil, and oregano, or their extracts; grains such as rice (indica, japonica, glutinous rice), cereals (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, yellow millet, buckwheat, soybean, broad bean, pea, mung bean, red bean, and kidney bean, or their extracts; nuts such as walnut, pistachio, cashew, hazelnut, almond, apricot kernel, pine nut, peanut, sunflower seed, chestnut, macadamia nut, and ginkgo, or their extracts; coffee or its extracts; and some medicinal and edible herbal medicines or their extracts.

[0103] Animal dairy product ingredients can include fresh milk from cows and sheep, as well as reprocessed dairy products such as whole milk powder, skim milk powder, whey protein concentrate, desalted whey powder, whey protein powder, hydrolyzed whey protein powder, and casein powder.

[0104] Animal meat product ingredients can include pork, beef, mutton, seafood, or poultry meat products.

[0105] Functional additives can include vitamin supplements (such as vitamin A, beta-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, folic acid, niacin, biotin, etc.), mineral supplements (such as iron, copper, manganese, zinc, cobalt, molybdenum, chromium, nickel, vanadium, fluorine, selenium, iodine, silicon, tin, etc.), nucleotide supplements (e.g.), dietary fiber (e.g., inulin, konjac flour, galactooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin, soybean fiber, etc.), and functional polyunsaturated fatty acid supplements (e.g., arachidonic acid oil powder, docosahexaenoic acid oil powder, etc.).

[0106] Any acceptable excipients may include solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, food flavorings, food colorings, etc.

[0107] This invention demonstrates through experimental data that the obtained corn extract exhibits a significant dose-dependent GLP-1 secretion-promoting effect, effectively increasing GLP-1 secretion levels in L cells under normal physiological conditions, confirming its high efficiency and mechanism of action. More importantly, this invention evaluates its efficacy in a palmitic acid-induced insulin resistance model of L cells (such as STC-1 cells), a model that more accurately simulates the cellular environment under the pathological state of type II diabetes. Compared to most existing technologies that are only tested in healthy cell models, this evaluation system is more pathologically relevant, and the conclusions obtained provide more convincing experimental evidence and prospective value for subsequent clinical application translation.

[0108] The embodiments and experimental examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0109] The materials and reagents used in the experiments described in this example and their sources are as follows: Fresh yellow sweet corn was purchased from Heilongjiang North Latitude 47 Green Organic Food Co., Ltd.; mouse glucagon-like peptide-1 (GLP-1) ELISA kit and mouse cyclic adenosine monophosphate (cAMP) ELISA kit were purchased from Shanghai Future Industrial Co., Ltd.; MTT cell proliferation and cytotoxicity assay kit and Fluo-4 calcium ion assay kit were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; RNA extraction kit was purchased from Guangzhou Meiji Biotechnology Co., Ltd.; and reverse transcription and PCR kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0110] Unless otherwise specified in the examples, the conditions shall be performed under standard conditions or conditions recommended by the manufacturer. Unless otherwise specified, all materials and instruments used shall be commercially available products.

[0111] Example 1: Preparation of corn extract

[0112] (1) Fresh corn (Overland) was selected and damaged kernels were removed. The kernels were then quickly peeled off and thoroughly rinsed with running deionized water to remove surface impurities and visible dust. The processed corn kernels were placed in a freeze dryer and freeze-dried under vacuum for 48-72 hours until the moisture was almost completely removed, resulting in a loosely textured freeze-dried corn product.

[0113] (2) The freeze-dried corn kernels were first pre-crushed using a high-speed pulverizer, and then further processed by ball milling to make the powder fine and uniform. The crushed sample was sieved through a 60-mesh sieve to remove larger uncrushed particles and obtain uniform fine powder. The sieved powder was placed in a desiccator for equilibration for 30 min.

[0114] (3) Weigh a certain amount of corn powder and add 100% ethanol solution at a material-to-liquid ratio of 1:10 (m / v). To promote the full penetration of ethanol into the powder, the mixture is first gently shaken and pre-soaked at room temperature for 20 min. Then, the system is placed in an ultrasonic cell disruptor and ultrasonicated at 40℃ and 100W power for 60 min, with a pulse of 10 s and a pause of 2 s. After extraction, centrifuge at 10000rpm for 10 min and collect the first supernatant.

[0115] (4) The precipitate obtained by centrifugation was added again with 100% fresh ethanol at a material-to-liquid ratio of 1:10 (m / v), and the above pre-soaking, ultrasonic extraction, and centrifugation steps were repeated twice to maximize the extraction efficiency of soluble components. After combining all supernatants, if suspended particles were present, they were filtered through a 0.45 μm filter membrane. The filtrate was then concentrated under reduced pressure in a rotary evaporator at 40°C in a water bath until most of the ethanol was recovered and the extract was concentrated.

[0116] (5) The concentrated extract was frozen at -80°C for several hours until completely solidified, and then placed in a freeze dryer for 48 h of vacuum freeze drying to finally obtain a loose corn crude extract powder. The obtained freeze-dried powder was crushed and sealed in a brown bottle and stored at -20°C in the dark for subsequent bioactivity screening or component analysis.

[0117] Example 2: The preparation and extraction method is the same as in Example 1, except that the corn variety is changed to Shengtian 1365.

[0118] Example 3: The preparation and extraction method is the same as in Example 1, except that the corn variety is changed to SBS903.

[0119] Reference Example 1: The preparation and extraction method is the same as in Example 1, except that the corn variety is changed to YSC106.

[0120] Reference Example 2: The preparation and extraction method is the same as in Example 1, except that the corn variety is changed to YSC113.

[0121] Reference Example 3: The preparation and extraction method is the same as in Example 1, except that the corn variety is changed to Crispy King.

[0122] Experimental Example 1: STC-1 Cytotoxicity Detection Assay

[0123] Experimental methods:

[0124] Cell culture: Mouse small intestinal endocrine cells (STC-1 cells) were cultured in high-glucose DMEM medium supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin under the following conditions: 5% carbon dioxide and 37°C incubator. When the cell confluence reached 80%-90%, the cells were passaged.

[0125] Sample preparation: 0.8 g of freeze-dried crude corn extract powder prepared in Example 1 was dissolved in 1 mL of DMSO to obtain a stock solution with a concentration of 800 mg / mL. The solution was diluted with cell culture medium to make the final concentration of corn extract in contact with the cells 50, 100, 200, 400, 600, 800, and 1000 μg / mL.

[0126] Cell model establishment: STC-1 cells were used at a rate of 1×10⁻⁶. 5 STC-1 cells were seeded at a density of cells / mL in 96-well plates and cultured for 24 h. Then, different concentrations of diluted maize extract samples were added to STC-1 cells for 24 h of intervention treatment, followed by 0.4 mM palmitic acid for 24 h of intervention treatment.

[0127] Cell viability was assessed using the Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. 10 μL of MTT solution was added to each well of cells treated with palmitic acid, and the cells were incubated at 37°C for 4 h. Subsequently, 100 μL of Formazan solution was added to each well to dissolve the purple crystals. After gentle vortexing, the absorbance was measured at 570 nm using a microplate reader.

[0128] Experimental results:

[0129] Palmitic acid levels are significantly elevated in the blood of patients with type 2 diabetes and obesity, thus it can highly mimic the pathological state. Therefore, palmitic acid intervention was used to treat STC-1 cells to simulate an insulin resistance model. Figure 1 As shown, the effect of different concentrations of palmitic acid on the viability of STC-1 cells was first examined (without corn extract intervention). The results showed that the cytotoxicity induced by palmitic acid increased in a dose-dependent manner, inducing 43% cell death at 0.4 mM, which can be used as a dose concentration for subsequent simulation of insulin resistance. Figure 2 As shown, corn extract at doses of 50-1000 μg / mL was used to intervene in cells (without palmitic acid intervention). It was found that 1000 μg / mL of corn extract was toxic to cell viability. Therefore, the concentrations of corn extract used to intervene in STC-1 cells were subsequently selected as 100, 200, 400, 600, and 800 μg / mL.

[0130] To investigate whether corn extract intervention could protect against, or even reverse, palmitic acid-induced STC-1 cell damage, cells were protected with corn extract at doses of 100-800 μg / mL. After 24 h, STC-1 cells were treated with 0.4 mM palmitic acid to induce damage, and changes in cell viability were then observed. Figure 3 As shown, compared with the palmitic acid group, corn extract has a significant protective effect against palmitic acid-induced cell damage, and the protective effect is dose-dependent. 800 μg / mL of corn extract increased the survival rate of STC-1 cells by 41%.

[0131] Experiment Example 2: GLP-1 Secretion Detection Experiment in STC-1 Cells

[0132] Experimental methods:

[0133] Cell culture and sample preparation were the same as in Experiment 1, except that the samples also included corn extracts prepared in Examples 2-3 and Reference Examples 1-3.

[0134] Detection under normal cell conditions: STC-1 cells were cultured at 1×10⁻⁶ cells per cell. 5 STC-1 cells were seeded at a density of [number] cells / mL in 24-well plates and cultured for 24 h. Then, a series of corn extracts were added to the cells for intervention for 24 h. After intervention, the cells were washed three times with phosphate-buffered saline (PBS), and Krebs-Ringer buffer and 50 μM MDPP4 inhibitor were added. The cells were incubated at 37°C and 5% CO2 for 2 h. After incubation, the cell supernatant was collected on ice and centrifuged at 4°C and 10,000 rpm for 10 min. The supernatant was then analyzed using a mouse glucagon-like peptide-1 (GLP-1) ELISA kit (Jianglai Biotechnology). Simultaneously, the protein content of each well was measured to normalize the cell count.

[0135] Detection in an insulin resistance model: STC-1 cells were loaded at a rate of 1×10⁻⁶. 5 STC-1 cells were seeded at a density of [number] cells / mL in 24-well plates and cultured for 24 h. Then, a series of treatments involving corn extract were added to the cells for 24 h, followed by palmitic acid treatment for another 24 h. After the intervention, the cells were washed three times with PBS, and Krebs-Ringer buffer and 50 μM DPP4 inhibitor were added. The cells were incubated at 37°C and 5% CO2 for 2 h. After incubation, the cell supernatant was collected on ice and centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was then analyzed using a mouse glucagon-like peptide-1 (GLP-1) ELISA kit (Jianglai Biotechnology). Simultaneously, the protein content of each well was measured to normalize the cell count.

[0136] Experimental results:

[0137] STC-1 cells under normal conditions were treated with different concentrations of the corn extract from Example 1, and the GLP-1 content in the cell supernatant was measured. Figure 4 As shown, compared with the control group (no corn extract intervention), different concentrations of the corn extract from Example 1 all induced significant dose-dependent GLP-1 release, indicating that under normal (healthy) physiological conditions, the corn extract from Example 1 has the ability to promote GLP-1 secretion. Figure 5 As shown, when STC-1 cells were treated with corn extracts of different varieties at 100 μg / mL, compared with other corn varieties in the reference examples, the corn extracts obtained in Examples 1-3 were able to more effectively stimulate the secretion of GLP-1 in STC-1 cells, and the resulting GLP-1 content was at a more significant level.

[0138] In addition, such as Figure 6 As shown, palmitic acid intervention significantly reduced GLP-1 secretion levels in STC-1 cells. When STC-1 cells were treated with different concentrations of the corn extract prepared in Example 1, it was found to significantly improve or even reverse the palmitic acid-induced decrease in GLP-1 secretion. Compared to the palmitic acid group, 800 μg / mL of the corn extract prepared in Example 1 increased GLP-1 secretion levels in STC-1 cells by 40%. Similarly, the corn extracts prepared in Examples 2-3 yielded similar experimental results to those obtained with the corn extract prepared in Example 1.

[0139] Experiment Example 3: Detection of Cyclic Adenosine Monophosphate in STC-1 Cells

[0140] Experimental methods:

[0141] Cell culture and sample preparation are the same as in Experiment 1.

[0142] STC-1 cells were loaded at 1×10 5 STC-1 cells were seeded at a density of [number] cells / mL in 24-well plates and cultured for 24 h. Then, a series of treatments involving corn extract were added to the cells for 24 h, followed by palmitic acid treatment for another 24 h. After the intervention, the cells were washed three times with PBS and incubated for 2 h in a 37°C, 5% CO2 incubator with Krebs-Ringer buffer. After incubation, the cell supernatant was collected on ice and centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was then analyzed using a mouse cyclic adenosine monophosphate (cAMP) ELISA kit (Jianglai Biotechnology). Simultaneously, the protein content of each well was measured to normalize the cell count.

[0143] Experimental results:

[0144] cAMP is produced from ATP catalyzed by adenylate cyclase (AC). Studies have shown that palmitic acid typically inhibits adenylate cyclase activity and activates PDE (phosphodiesterase), accelerating cAMP degradation. cAMP levels in STC-1 cells were detected using an ELISA kit. Figure 7 As shown, palmitic acid intervention significantly reduced the cAMP level in cells, while the corn extract prepared in Example 1 could increase the cAMP content in a dose-dependent manner. cAMP, as an intracellular second messenger, participates in the secretion of GLP-1 in cells.

[0145] Similarly, the corn extracts prepared in Examples 2-3 also yielded similar experimental results to those obtained in Example 1.

[0146] Experiment Example 4: Detection of Calcium Ions in STC-1 Cell Cytoplasm

[0147] Experimental methods:

[0148] Cell culture and sample preparation are the same as in Experiment 1.

[0149] Cytoplasmic calcium levels were detected using the Fluo-4 calcium ion assay kit. STC-1 cells were cultured at a concentration of 1 × 10⁻⁶ cells / mL. 5 STC-1 cells were seeded at a density of [number] cells / mL in 12-well plates and cultured for 24 h. Then, a series of treatments were initiated by adding corn extract to the cells for 24 h, followed by palmitic acid treatment for another 24 h. The culture medium was then aspirated, the cells were washed with PBS, and an appropriate volume of Fluo-4 staining solution was added. The cells were incubated at 37°C in the dark for 30 min. After incubation, the staining effect was observed under a fluorescence microscope (Fluo-4 AM is green fluorescence, Ex / Em = 490 / 525 nm). The fluorescence intensity was measured using a microplate reader.

[0150] Experimental results:

[0151] To investigate the intracellular mechanism of GLP-1 release induced by corn extract, the role of potential second messengers was analyzed in depth. Intracellular calcium ion fluctuations were measured using an intracellular calcium ion indicator. Figure 8 As shown, the fluorescence intensity in STC-1 cells treated with the corn extract prepared in Example 1 was enhanced. This enhanced fluorescence intensity directly reflects the increase in intracellular calcium ion concentration, suggesting that the corn extract prepared in Example 1 may bind to the SUR1 receptor, causing the KATP channel to close, thereby promoting the opening of calcium ion channels and ultimately triggering calcium ion influx. GLP-1 secretion is positively correlated with intracellular calcium ion levels. Figure 8As shown, palmitic acid intervention significantly reduced the cytoplasmic calcium level of STC-1 cells, and the cytoplasmic calcium level was improved after treatment with the corn extract prepared in Example 1.

[0152] Similarly, the corn extracts prepared in Examples 2-3 also yielded similar experimental results to those obtained in Example 1.

[0153] Experiment Example 5: Detection of GLP-1 secretion-related genes

[0154] Experimental methods:

[0155] Cell culture and sample preparation are the same as in Experiment 1.

[0156] STC-1 cells were loaded at 1×10 5STC-1 cells were seeded at a density of [number] cells / mL in 12-well plates and cultured for 24 h. Then, a series of treatments involving corn extract were added to the cells for 24 h, followed by palmitic acid treatment for another 24 h. After the intervention, cellular RNA was extracted using a total RNA extraction kit. After the intervention, the culture medium was completely aspirated, and an appropriate amount of RTL Lysis Buffer was added to each well to disperse the cells. The cells were then transferred to 1.5 mL centrifuge tubes, and the lysis buffer was pumped 5-10 times to fragment the DNA. An equal volume of RNA Binding Buffer was added to the lysis buffer, and the cells were pumped 3-10 times. The mixture was transferred to a HiPure RNA Mini Column, centrifuged at 12000×g for 30-60 s, the filtrate was discarded, and 300 μL of Buffer RW1 was added to the column. The column was centrifuged at 12000×g for 2 min. DNase working solution was added to the column, and the column was incubated at room temperature for 20-30 min. Add 500 μL Buffer RW1 to the column, incubate for 3 min, centrifuge at 12000×g for 30-60 s, discard the filtrate, add 500 μL Buffer RW2 to the column, centrifuge at 12000×g for 30-60 s, repeat once, discard the filtrate, transfer the column back to the collection tube, centrifuge at 12000×g for 2 min, transfer the column to a 1.5 mL centrifuge tube, add 20 μL RNase-free water to the center of the column membrane, incubate at room temperature for 2 min, centrifuge at 12000×g for 1 min, discard the column, and store the RNA at -80℃. Use a reverse transcriptase kit to synthesize cDNA from the mRNA in each group of samples. The synthesized cDNA was then amplified and detected using a real-time quantitative PCR (RT-qPCR) instrument. The PCR parameters were: 50 °C for 2 min, 95 °C pre-denaturation for 10 min, 95 °C denaturation for 15 s, and 60 °C amplification for 1 min, for 40 cycles. Changes in target gene expression were expressed as relative mRNA levels and normalized to GAPDH. Data were analyzed using a 2-1... -ΔΔCt Calculation and analysis.

[0157] The primer sequences for each gene are shown in Table 1 below.

[0158] Table 1 Primer sequences of genes

[0159]

[0160] Experimental results:

[0161] The proglucagon precursor gene (Gcg) encodes GLP-1, and all forms of GLP-1 are produced from proglucagon. The precursor protein convertase gene (Pcsk1) converts proglucagon into GLP-1. This study investigates whether palmitic acid intervention in maize extract increases the expression of genes related to GLP-1 secretion by detecting the expression levels of these two genes. Figure 9 As shown, palmitic acid intervention significantly downregulated the expression levels of Gcg and Pcsk1 genes, while treatment with the corn extract prepared in Example 1 significantly increased the expression of both genes. The maximum dose of corn extract (800 μg / mL) increased the expression of Gcg and Pcsk1 genes by 6.94 and 4.92 times, respectively.

[0162] Similarly, the corn extracts prepared in Examples 2-3 also yielded similar experimental results to those obtained in Example 1.

[0163] Experiment Example 6: Detection of GLP-1 secretion-related proteins

[0164] Experimental methods:

[0165] Cell culture and sample preparation are the same as in Experiment 1.

[0166] STC-1 cells were used at a rate of 1×10 5STC-1 cells were seeded at a density of [number] cells / mL and cultured in 12-well plates for 24 h. A series of treatments were then initiated with corn extract for 24 h, followed by palmitic acid treatment for another 24 h. After intervention, the supernatant was removed, washed three times with PBS, and placed on ice. Cells were lysed for 30 min on ice using RIPA buffer containing 1% protease inhibitor and 10% phosphatase inhibitor, with vortexing every 10 min to ensure complete lysis. After centrifugation at 12000 rpm for 10 min at 4 °C, the supernatant was removed and placed on ice. The protein content in the supernatant was measured using a BCA protein assay kit, and the protein content was normalized with PBS. Denaturing agent was added to the supernatant at a 1:3 volume ratio, and the mixture was vortexed and denatured in a 95 °C metal bath for 5 min. The supernatant was then stored at -20 °C for subsequent Western blotting. Protein samples were separated using a 12% polyacrylamide gel, with 10 μL of protein sample added to each lane and markers applied at both ends to distinguish different protein molecular weights. The target proteins were separated using an electrophoresis program of 80 V × 20 min, then transferred to 120 V × 50 min using a transfer apparatus at 20 V, 1 A onto a nitrocellulose (NC) membrane. The membrane was blocked with 5% skim milk in 0.05% Tween-20 wash buffer (TBST) at room temperature for 1 h, followed by three washes with TBST. Diluted p-PKA, PKA, p-CaMKII, CaMKII, p-ERK, ERK, CREB, p-CREB, and α-tubulin antibodies were added, and the membrane was incubated overnight at 4 °C with shaking. After incubation with secondary antibodies at room temperature for 1.5 h, the membrane was analyzed and photographed using a chemiluminescence method on a Bio-Rad imaging system. The grayscale values ​​of the protein bands were quantified using Image Lab software.

[0167] Experimental results:

[0168] This experiment investigated the potential signaling pathways by which corn extract promotes GLP-1 release from STC-1 cells. These signaling pathways include protein kinase A (PKA), calmodulin-dependent protein kinase 2 (CaMKII), exogenously regulated protein kinase (ERK), and cAMP response element-binding protein (CREB). To assess the activation of these pathways, Western blot was used to detect the effects of corn extract on the phosphorylation levels of PKA and CaMKII and the expression of downstream proteins. Figure 10As shown, palmitic acid intervention significantly inhibited the phosphorylation levels of PKA, CaMKII, ERK, and CREB proteins, resulting in a 53%, 42%, 56%, and 49% reduction in the protein expression of p-PKA, p-CaMKII, p-ERK, and p-CREB, respectively. In contrast, the corn extract prepared in Example 1 increased the phosphorylation levels of PKA, CaMKII, ERK, and CREB proteins in a dose-dependent manner, suggesting that the above signaling pathways may synergistically participate in the corn extract-promoted GLP-1 secretion.

[0169] Similarly, the corn extracts prepared in Examples 2-3 also yielded similar experimental results to those obtained in Example 1.

Claims

1. A corn extract that helps promote GLP-1 secretion, characterized in that, The corn extract is a dried product obtained by ultrasonic extraction of dried powder of fresh corn kernels after soaking in 100% ethanol at room temperature; the fresh corn includes yellow sweet corn; the ultrasonic extraction temperature is 30~60℃; the ultrasonic extraction power is 50~150W; the ultrasonic extraction time is 30~90min; the yellow sweet corn variety includes at least one of Auferan, Saint Sweet 1365 and SBS903.

2. The corn extract according to claim 1, characterized in that, The dried powder of fresh corn kernels is prepared by the following steps: take fresh corn kernels, wash them, freeze-dry them under vacuum, pulverize them, and pass them through a 40-80 mesh sieve. Place the sieved powder in a desiccator to balance it, and you will get the powder.

3. The corn extract according to claim 1 or 2, characterized in that, The soaking time at room temperature is 15-30 minutes.

4. The method for preparing corn extract according to any one of claims 1 to 3, characterized in that, The method includes the following steps: taking fresh corn kernels, making them into a dry powder, weighing the powder and mixing it with ethanol at a mass-volume ratio of (0.5-5):10, soaking it at room temperature and then performing ultrasonic treatment, separating the solid and liquid to obtain a supernatant and a precipitate, repeating the process of mixing with ethanol at a mass-volume ratio of (0.5-5):10, soaking at room temperature and then performing ultrasonic treatment and solid-liquid separation on the precipitate once or twice or more, combining all the obtained supernatants and performing vacuum concentration and freeze drying to obtain the final product.

5. The method according to claim 4, characterized in that, The vacuum concentration is carried out using a rotary evaporator under water bath conditions of 35~45℃.

6. Use of the corn extract according to any one of claims 1 to 3 in the preparation of products that help maintain healthy blood sugar levels; The benefits of maintaining healthy blood sugar levels include those that help maintain healthy blood sugar levels even in a state of insulin resistance.

7. The use according to claim 6, characterized in that, The products include food.

Images