A low-glucose-modulating camelina sativa seed meal oligosaccharide extract, a preparation method thereof, a blood glucose-lowering composition, and an application thereof

Abstract

The application discloses a kind of with regulating blood sugar camelia seed meal oligosaccharide extract and preparation method thereof, utilize fermentation, heating extraction, cooling alcohol precipitation, enzyme hydrolysis from camelia seed meal and extract oligosaccharide extract, the application also discloses a kind of hypoglycemic composition, including camelia seed meal oligosaccharide extract and aesculetin class component extract;Aesculetin class component extract is extracted by microwave-assisted enzyme hydrolysis, and is obtained by purification;The application also discloses the application of the hypoglycemic composition in hypoglycemic food, drug or health care product.The camelia seed meal oligosaccharide extract, aesculetin class component extract and hypoglycemic composition described in the application have inhibiting capacity to α-glucosidase and α-amylase, can improve glucose consumption, glycogen content and glycolysis key enzyme activity, effectively improve insulin resistance, improve oxidative stress, regulate intestinal flora structure, safe and non-toxic, and have good efficacy for treating diabetes and complications.

Description

Technical Field

[0001] This invention belongs to the field of bio-extraction technology, specifically relating to a blood sugar-regulating flaxseed meal oligosaccharide extract, its preparation method, and its application in blood sugar-regulating products. It also relates to a blood sugar-lowering composition containing the flaxseed meal oligosaccharide extract and its application in blood sugar-lowering foods, medicines, or health products. Background Technology

[0002] Diabetes mellitus (DM) is a chronic metabolic disease characterized primarily by hyperglycemia. Long-term hyperglycemia can damage multiple systems and organs throughout the body, leading to chronic complications such as heart disease, stroke, kidney failure, blindness, neuropathy, and diabetic foot, severely impacting quality of life and even endangering life. As a public health problem that seriously threatens human health after cancer and cardiovascular diseases, diabetes's incidence and mortality rates are rapidly increasing worldwide. China, with the largest number of diabetes patients in the world, has seen increasing attention from researchers regarding the prevention and treatment of diabetes.

[0003] Currently, clinical treatment of diabetes primarily relies on insulin and its analogues, biguanides, sulfonylureas, meglitinides, thiazolidinediones, and dipeptidyl peptidase-4 (DPP-4) inhibitors. While these drugs are effective, they are accompanied by adverse reactions such as gastrointestinal discomfort, hypoglycemia, and weight gain. Long-term use may lead to liver and kidney damage. Therefore, developing a safe, effective, and low-side-effect hypoglycemic drug with minimal burden on liver and kidney function is of significant practical importance.

[0004] Flaxseed (Capsella bursa-pastoris) is an annual herbaceous oilseed crop belonging to the genus Capsella of the Brassicaceae family. It has a long history of cultivation and is distributed in Xinjiang, Heilongjiang, Henan, Shaanxi, and other regions of my country. Both flaxseed and the plant contain polysaccharides, polyphenols, and potential functional components. The literature "Response Surface Optimization of Flaxseed Polysaccharide Extraction Process and Its In Vitro Antioxidant Activity" describes the extraction process and antioxidant activity of crude flaxseed polysaccharides. Patent document CN118948699A discloses a method for obtaining a flaxseed polysaccharide extract with anti-inflammatory and antioxidant effects through water extraction, followed by fermentation, enzymatic hydrolysis, alcohol precipitation, ultrafiltration, and resin column elution. However, flaxseed polysaccharides, as large molecules, have high viscosity and complex structures, which hinders absorption and utilization by the body, preventing them from effectively exerting their biological activity and greatly limiting their application. Flaxseed meal, a byproduct of flaxseed oil extraction, is typically used as animal feed or fertilizer, with low economic added value. Reports on deep processing technologies for flaxseed meal are scarce, with current processing focusing on further oil extraction, value-added utilization of residual oils, and assessment of its feed value. To enhance the high-value utilization of flaxseed meal, further research is needed to develop extraction methods and effective utilization of functional components.

[0005] Methods for extracting carbohydrates from plant seed meals include hot water extraction, acid-base extraction, fermentation, enzymatic hydrolysis, and physical aids such as microwave and ultrasound. CN201510830656.0 discloses a tea seed oligosaccharide, the preparation steps of which include reflux extraction of defatted tea seed meal with an ethanol-water solution, adding water to the filter residue after removing ethanol, adding enzyme preparation 1 for enzymatic hydrolysis, filtering, obtaining the filtrate, treating with enzyme preparation 2, then ultrafiltration, macroporous resin adsorption, ethanol-water desorption, vacuum concentration and drying to obtain the tea seed oligosaccharide. The main component of tea seed meal is tea saponin (water-soluble glycosides), which can be degraded into small molecule sugars through enzymatic hydrolysis. However, the characteristics and composition of flaxseed meal differ substantially from those of tea seed meal. The main components of flaxseed meal are protein and anti-nutritional factors (glucosinolates, phytic acid, cellulose, lectins, and trypsin inhibitors, etc.). These anti-nutritional factors interfere with the extraction of flaxseed meal (glucosinolate enzymatic hydrolysis produces toxic substances), increasing the difficulty of preparation. CN201910098976.X discloses a functional mulberry leaf oligosaccharide and its preparation method, which is obtained by defatting, water extraction, enzymatic hydrolysis, alcohol precipitation and fermentation. Although the main components of mulberry leaves are protein and cellulose, this method is used for the extraction of flaxseed meal, resulting in low yield and low product purity.

[0006] In addition, Fraxinus chinensis bark is cold in nature and bitter in taste, possessing the effects of clearing heat and drying dampness, astringing, and improving eyesight. Modern research has found that iridoid ethers in Fraxinus chinensis bark are one of its important active ingredients. Currently, pharmacological activity studies of flaxseed and Fraxinus chinensis bark mainly focus on their antioxidant, anti-inflammatory, and antibacterial properties, but there are no related studies or reports on the hypoglycemic effects of flaxseed extract combined with Fraxinus chinensis bark extract. This invention delves into the hypoglycemic effects and mechanisms of flaxseed meal oligosaccharide extract, Fraxinus chinensis bark iridoid ether extract, and their combination in the treatment of diabetes, aiming to provide a theoretical basis for the development of hypoglycemic-related products. Summary of the Invention

[0007] To enhance the high-value utilization of flaxseed meal, this invention aims to provide a flaxseed meal oligosaccharide extract with blood sugar regulating properties and its preparation method. The oligosaccharide extract is obtained from flaxseed meal through fermentation, heating extraction, cooling alcohol precipitation, enzymatic hydrolysis, and dialysis, resulting in detoxification and high yield. This invention also discloses the application of this flaxseed meal oligosaccharide extract in blood sugar regulating products.

[0008] To address the toxic side effects of hypoglycemic drugs, another objective of this invention is to provide a hypoglycemic composition containing the aforementioned flaxseed meal oligosaccharide extract. The flaxseed meal oligosaccharide extract, in conjunction with the fraxinus erythrolene ether extract, significantly enhances the hypoglycemic effect without toxic side effects. This invention also discloses the application of this hypoglycemic composition in hypoglycemic foods, pharmaceuticals, or health products.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A method for preparing a flaxseed meal oligosaccharide extract with blood sugar regulating properties includes the following steps:

[0011] S1. Flaxseed meal is dried, pulverized, and sterilized, and used as the extraction raw material. The extraction raw material is mixed evenly with an aqueous solution containing fermentation bacteria at a mass ratio of 1:4-7, and fermented at 25-35℃ for 24-72 hours to obtain the fermentation product; wherein the concentration of fermentation bacteria in the aqueous solution containing fermentation bacteria is 3×10⁻⁶. 6 -6×10 7 CFU / mL;

[0012] S2. Add water to dilute the fermentation product obtained in step S1, and extract it by stirring at a constant temperature of 85-100℃ for 1-3 hours under normal pressure and closed environment. Then, separate the solid and liquid, take the liquid, concentrate it to a mass-volume fraction of 0.3-0.5 kg / L, and separate the supernatant.

[0013] S3. Add ethanol or an aqueous ethanol solution to the product obtained in step S2, mix well, and obtain a mixed solution containing 75-85 vol% ethanol. Let the mixed solution stand at 3-6℃ for 20-30 h, separate the solid and liquid, take the solid, add water to dissolve, and obtain an aqueous extract solution.

[0014] S4. Add enzyme to the aqueous extract obtained in step S3, heat to 40-60℃ for 3-6 hours for enzymatic hydrolysis, then inactivate the enzyme, separate the solid and liquid, take the liquid, and after dialysis, concentrate and dry to obtain flaxseed meal oligosaccharide extract; wherein, the amount of enzyme added is 2-5% of the mass of the raw material extracted in step S1.

[0015] This invention utilizes fermentation to disrupt the cell wall structure of raw materials, promoting the release of active ingredients; heating to enhance extraction efficiency; alcohol precipitation to separate impurities; and finally, enzymatic hydrolysis to specifically degrade non-target polysaccharides or impurities, significantly improving the yield and purity of oligosaccharides. By employing fermentation and enzymatic hydrolysis as the core processing methods, it reduces the use of organic solvents, lowers environmental pollution and subsequent processing costs, and avoids the damage to oligosaccharide structures caused by high temperatures or chemical treatments, while also saving energy. This invention reduces intermediate product processing steps, lowers operational complexity and production costs, and improves the utilization rate of flaxseed meal by sequentially processing flaxseed meal through fermentation, heating extraction, cooling alcohol precipitation, and enzymatic hydrolysis.

[0016] To efficiently decompose proteins, the preparation steps of the aqueous solution containing fermenting bacteria are as follows: Take *Saccharomyces cerevisiae* strain as the inoculum to be activated, dilute it with sterile water or glucose liquid medium, and inoculate it onto glucose solid medium. Incubate at 25-35℃ for 48-72 hours. Then, use an inoculation loop to pick mature, single colonies and inoculate them into glucose liquid medium. Incubate at 25-35℃ with shaking for 24-30 hours, then adjust the bacterial concentration to 1×10⁻⁶. 8 -1×10 9 A fermentation culture suspension was obtained by dispersing CFU / mL of the culture medium. The suspension was then diluted with water to obtain an aqueous solution containing the fermentation bacteria. The glucose solid culture medium (g / L) contained: 5-7 g / L peptone, 7-10 g / L beef extract, 5-10 g / L glucose, 4-5 g / L sodium chloride, and 15-18 g / L agar. After preparation according to the specified ratio, the medium was sterilized at 121°C for 20 min, poured into petri dishes, and allowed to solidify at room temperature to obtain the glucose solid culture medium. The glucose liquid culture medium (g / L) contained: 5-7 g / L peptone, 7-10 g / L beef extract, 5-10 g / L glucose, and 4-5 g / L sodium chloride. After preparation according to the specified ratio, the medium was sterilized at 121°C for 20 min and allowed to solidify at room temperature to obtain the glucose liquid culture medium.

[0017] To further improve the extraction rate of the target component (oligosaccharides), the enzyme in step S4 is selected from cellulase, pectinase or xylanase.

[0018] To ensure extraction effectiveness and improve extraction efficiency, in step S2, the amount of water used for dilution is 8-12 times the weight of the raw material to be extracted; in step S3, the volume fraction of ethanol in the ethanol-water solution is 90-95%; and in step S3, the amount of water used for dissolving is 1-1.5 times the mass of the raw material to be extracted in step S1.

[0019] To ensure the purity of the extract, pretreatment, intermediate separation, and post-treatment are necessary. Sterilization in step S1 is performed at 0.08-0.12 MPa and 120-125℃ for 15-25 minutes. Separation of the supernatant in step S2 and solid-liquid separation in step S3 are both performed by centrifugation, with a centrifugation speed of 4500-5500 rpm and a centrifugation time of 8-12 minutes. Solid-liquid separation in steps S2 and S4 is performed by filtration, and concentration in steps S2 and S4 is performed by vacuum concentration. Enzyme inactivation in step S4 is performed by boiling in a water bath for 4-6 minutes. The dialysis process involves dialyzing the liquid obtained from solid-liquid separation using a dialysis bag with a molecular weight of 300 Da, changing the water at 8h, 12h, and 16h, dialyzing for 24h, collecting the retained solution, and then dialyzing again using a dialysis bag with a molecular weight of 2000 Da for 24h, collecting the permeate.

[0020] Preferably, the drying in step S4 is freeze drying, and the freeze drying conditions are: vacuum degree 10-50 Pa, temperature -20 to 40℃, and duration 24-48 h.

[0021] The oligosaccharide extract of flaxseed meal prepared by the above method has an oligosaccharide content of over 92% and a molecular weight of 300-2000 Da.

[0022] A hypoglycemic composition comprising the following raw materials in parts by weight: 4-9 parts of the flaxseed meal oligosaccharide extract and 3-6 parts of the fraxinus chinensis cyclohexene ether terpene extract.

[0023] The extract of cyclohexene ethers and terpenoids from Fraxinus chinensis was prepared using the following steps: Crushed and sieved Fraxinus chinensis was dispersed in DES aqueous solution at a material-to-liquid ratio of 1:(20-40) g / mL to obtain a mixed solution; cellulase was added to the mixed solution, and after mixing, it was microwave extracted at 40-60℃ for 30-60 min; the solution was filtered, the filtrate was collected, and concentrated to a mass-to-volume fraction of 0.3-0.5 kg / L; the supernatant was separated and injected into an adsorption resin; the resin was first eluted with water until the eluent was nearly colorless, the eluent was discarded, and then the resin was extracted with 20-40 v... The product is eluted with 1% ethanol aqueous solution to remove impurities, the eluent is discarded, and finally eluted with 80-90 vol% ethanol aqueous solution. The eluent of 80-90 vol% ethanol aqueous solution is collected, concentrated under reduced pressure, and freeze-dried to obtain the final product. The DES aqueous solution is prepared by mixing DES solvent and water, with the water content in the DES aqueous solution being 10-30% by mass. The DES solvent is prepared by mixing choline chloride and carboxylic acid or choline chloride and polyol at a molar ratio of 1:2-3. The amount of cellulase added is 2-5% of the mass of Fraxinus chinensis bark.

[0024] Preferably, the carboxylic acid is oxalic acid, and the polyol is 1,4-butanediol or glycerol.

[0025] Preferably, during microwave extraction, the microwave power is 450-550W, and the supernatant is separated by centrifugation, with the centrifugation speed controlled at 4500-5500 rpm and the centrifugation time at 8-12 min.

[0026] Preferably, the freeze-drying conditions are: vacuum degree 10-50 Pa, temperature -20 to 40°C, and duration 24-48 h.

[0027] Preferably, the adsorption resin is a macroporous resin of type AB-8, D101, or NKA-9, and the amount of adsorption resin used is 1-1.5 times the mass of the Fraxinus bark; when eluting with water, the amount of water used is 5-8 times the mass of the Fraxinus bark, and the elution rate is 1.0-2.0 Bv / h; when eluting with 20-40 vol% ethanol aqueous solution, the amount of 20-40 vol% ethanol aqueous solution used is 5-8 times the mass of the Fraxinus bark, and the elution rate is 1.0-2.0 Bv / h; when eluting with 80-90 vol% ethanol aqueous solution, the amount of 80-90 vol% ethanol aqueous solution used is 9-12 times the mass of the Fraxinus bark, and the elution rate is 1.0-2.0 Bv / h.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] This invention employs fermentation, alcohol precipitation, and enzymatic hydrolysis to extract oligosaccharides from flaxseed meal. Fermentation disrupts the cell wall structure of flaxseed meal, hydrolyzes complex polysaccharides (such as cellulose and hemicellulose) into oligosaccharide precursors, and releases bound sugars. Water extraction is then used to extract the soluble sugars and other water-soluble components. Alcohol precipitation yields the sugar precipitate, followed by enzymatic hydrolysis to further decompose any remaining polysaccharides or oligomers. Finally, dialysis ensures that the molecular weight of the oligosaccharides is controlled within the ideal range. The selected yeast strain for fermentation is a specific dominant strain with advantages such as a powerful enzyme system, efficient material conversion ability, and broad substrate adaptability. Although fermentation, as the first step, may introduce impurities and increase the complexity of the reaction, the enzymes or sugars produced can effectively improve the yield and content of oligosaccharides in shepherd's purse seed meal. At the same time, during metabolism by the complex enzyme system of the microorganisms, not only are polysaccharides broken down into oligosaccharides that are more easily absorbed and utilized by the human body, but the introduction of functional groups or surface modification may also enhance biological activity, thereby significantly inhibiting α-glucosidase and α-amylase. Furthermore, after enzyme release and alcohol precipitation, no impurities are introduced, and the remaining polysaccharides can be specifically hydrolyzed, thereby further ensuring the yield and purity of oligosaccharides.

[0030] The flaxseed meal oligosaccharide extract and Fraxinus chinensis iridoid extract described in this invention are refined from flaxseed meal and Fraxinus chinensis bark. They exhibit significant inhibitory activity against α-glucosidase and α-amylase. Furthermore, they effectively alleviate insulin resistance in HepG2 cells induced by high glucose and insulin by significantly increasing glucose consumption, glycogen content, and the activity of key glycolytic enzymes. Animal model experiments have verified that the combined flaxseed meal oligosaccharide extract and Fraxinus chinensis iridoid extract effectively reduce blood glucose levels in diabetic mice, improve insulin resistance, alleviate oxidative stress, and regulate gut microbiota structure. In addition, the composition of this invention can also improve blood lipid levels and liver and kidney damage in diabetic mice, demonstrating good efficacy in treating diabetes and its complications.

[0031] The oligosaccharide extract from flaxseed meal obtained by this invention has a yield of over 4.65% and a content of over 92%; the extract of fraxinus terpenoids obtained has a yield of over 3.20% and a content of over 82%. The combination of different extracts exhibits excellent synergistic effects in both in vitro and in vivo hypoglycemic activity, and can be applied to hypoglycemic foods, pharmaceuticals, or health products. Attached Figure Description

[0032] Figure 1 The graphs show the inhibitory effects (n=6) of the flaxseed meal oligosaccharide extract described in Example 2, the fraxinus erythrorhizon extract described in Example 5, and the hypoglycemic compositions described in Examples 7-9 on α-glucosidase.

[0033] Figure 2 The graphs show the inhibitory effects (n=6) of the flaxseed meal oligosaccharide extract described in Example 2, the fraxinus erythrorhizon extract described in Example 5, and the hypoglycemic compositions described in Examples 7-9 on α-amylase.

[0034] Figure 3 This is a bar chart showing the effects of the flaxseed meal oligosaccharide extract described in Example 2, the fraxinus chinensis iridoid extract described in Example 5, and the hypoglycemic compositions described in Examples 7-9 on glucose consumption in IR-HepG2 cells (n=6).

[0035] Figure 4 This is a bar chart showing the effects of the flaxseed meal oligosaccharide extract described in Example 2, the fraxinus erythrorhizon extract described in Example 5, and the hypoglycemic compositions described in Examples 7-9 on glycogen content in IR-HepG2 cells (n=6).

[0036] Figure 5 This is a bar chart showing the effects of the flaxseed meal oligosaccharide extract described in Example 2, the fraxinus chinensis iridoid extract described in Example 5, and the hypoglycemic compositions described in Examples 7-9 on the HK activity of IR-HepG2 cells (n=6).

[0037] Figure 6 This is a bar chart showing the effects of the flaxseed meal oligosaccharide extract described in Example 2, the fraxinus chinensis iridoid extract described in Example 5, and the hypoglycemic compositions described in Examples 7-9 on the PK activity of IR-HepG2 cells (n=6).

[0038] Figure 7 This is a graph showing the effect of the hypoglycemic composition described in Example 9 on fasting blood glucose in diabetic mice (n=6).

[0039] Figure 8This is a graph showing the effect of the hypoglycemic composition described in Example 9 on oral glucose tolerance in diabetic mice (n=6). Figure 8 A) and bar chart ( Figure 8 B);

[0040] Figure 9 This is a bar chart showing the effect of the hypoglycemic composition described in Example 9 on insulin in diabetic mice (n=6);

[0041] Figure 10 This is an analytical graph showing the effect of the hypoglycemic composition described in Example 9 on the gut microbiota of diabetic mice (n=3). Figure 10 A is the dilution curve. Figure 10 B is a Beta diversity analysis diagram. Figure 10 C represents the Alpha diversity analysis diagram. Figure 10 D is a distribution map of the gut microbiota at the phylum level in mice. Detailed Implementation

[0042] To make the technical objectives, technical solutions, and beneficial effects of the present invention clearer, the technical solutions of the present invention will be further described below in conjunction with specific embodiments. However, the embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in the field or the product manual shall apply.

[0043] The instruments and materials used in the following examples are all commercially available products. Some of the instruments used are listed below: ME204 0.0001 g electronic balance (Shanghai Mettler Toledo), AUW220D 0.0001 g electronic balance (Shimadzu, Japan), pipettes: 100 μL, 200 μL, 1000 μL (Eppendorf), FW-80 high-speed universal grinder (Beijing Yongguang), HH-6 digital display constant temperature water bath (Changzhou Zhiborui Instrument Manufacturing Co., Ltd.), LGJ-12 freeze dryer (Beijing Songyuan Huaxing), microwave extraction... The instruments used were MAS-I (Shanghai Xinyi Microwave Chemical Technology Co., Ltd.) and a TU1810 UV spectrophotometer (Beijing Purkinje). Some of the materials used are listed below: Flaxseed meal (source: obtained from the hydraulic pressing of flaxseed oil in our laboratory; the hydraulic pressing process was as follows: an appropriate amount of flaxseed was crushed in a high-speed grinder for 5 minutes, then pressed in a hydraulic oil press for 120 minutes, controlling the pressing temperature at 50℃ and the pressure at 70MPa). *Saccharomyces cerevisiae* (strain number CICC 33190) was purchased from the China Industrial Microbial Culture Collection Center. Cellulase (20000 U / g), pectinase (20000 U / g), and xylanase (300000 U / g) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0044] In the following examples, the fermentation suspension used to prepare the aqueous solution containing the fermentation bacteria was prepared using the following steps: *Saccharomyces cerevisiae* strain was taken as the strain to be activated. The strain was diluted with 1 mL of sterile water or glucose liquid medium and inoculated onto glucose solid medium. The culture was then incubated at 30°C for 60 h. Mature, single colonies were then picked using an inoculation loop and inoculated into glucose liquid medium. After shaking culture at 30°C for 28 h, the bacterial concentration was adjusted to 1.0 × 10⁻⁶ with water. 9 A fermentation suspension was obtained by mixing CFU / mL. The glucose solid medium (g / L) contained: 6 g / L peptone, 8.5 g / L beef extract, 7.5 g / L glucose, 4.5 g / L sodium chloride, and 16.5 g / L agar. After preparation according to the specified ratio, the mixture was sterilized at 121°C for 20 min, poured into petri dishes, and allowed to solidify at room temperature to obtain the glucose solid medium. The glucose liquid medium (g / L) contained: 6 g / L peptone, 8.5 g / L beef extract, 7.5 g / L glucose, and 4.5 g / L sodium chloride. After preparation according to the specified ratio, the mixture was sterilized at 121°C for 20 min and allowed to solidify at room temperature to obtain the glucose liquid medium.

[0045] In the following examples, the oligosaccharide content was determined using a UV spectrophotometer (anthrone sulfate method), and the iridoid content was determined according to the UV method for determining the total iridoid content in the literature "Study on the Quality Standard of Total Iridoid Glycosides in Jasmine".

[0046] A method for preparing a flaxseed meal oligosaccharide extract with blood sugar regulating properties includes the following steps:

[0047] S1. Thoroughly dry, crush, sieve, and sterilize the flaxseed meal as the extraction raw material. Mix the extraction raw material with an aqueous solution containing fermentation bacteria at a mass ratio of 1:4-7, and ferment at 25-35℃ for 24-72 hours to obtain the fermentation product.

[0048] The sterilization is carried out at 0.08-0.12 MPa and 120-125℃ for 15-25 minutes; the aqueous solution containing fermentation bacteria is obtained by diluting the fermentation bacteria suspension with water, and the volume fraction of the fermentation bacteria suspension in the aqueous solution is 3-6% (i.e., the concentration of fermentation bacteria in the aqueous solution is 3×10⁻⁶). 7 -6×10 7 (CFU / mL)

[0049] S2. Add 8-12 times the weight of water to the fermentation product obtained in step S1 to dilute it, and extract it at a constant temperature of 85-100℃ for 1-3 hours. Then filter it, take the filtrate, concentrate it under reduced pressure to a mass-volume fraction of 0.3-0.5 kg / L, stop the concentration, centrifuge and separate the supernatant.

[0050] The centrifugation control is as follows: rotation speed of 4500-5500 rpm and duration of 8-12 min;

[0051] S3. Add 90-95 vol% ethanol aqueous solution to the product obtained in step S2, mix well, and obtain a mixed solution containing 75-85 vol% ethanol. Let the mixed solution stand at 3-6℃ for 20-30 h, centrifuge, and obtain a precipitate. Add water to the precipitate to dissolve it and obtain an aqueous extract solution.

[0052] The centrifugation control is as follows: the rotation speed is 4500-5500 rpm and the duration is 8-12 min; when adding water to dissolve, the amount of water used is 1-1.5 times the mass of the extracted raw material in step S1.

[0053] S4. Add enzyme to the aqueous extract obtained in step S3, heat to 40-60℃ for enzymatic hydrolysis for 3-6 hours, then inactivate the enzyme in a boiling water bath for 4-6 minutes, separate the solid and liquid, and collect the liquid to obtain the enzymatic extract; then, perform dialysis treatment (the temperature of the dialysis environment described below is guaranteed not to exceed 15℃): first, place the enzymatic extract in a dialysis bag with a molecular weight of 300 Da, change the water 3 times at 8h, 12h and 16h respectively, and dialyze for a total of 24h, and collect the retaining liquid; then place the retaining liquid in a dialysis bag with a molecular weight of 2000 Da, dialyze for 24h, and collect the permeate; concentrate the permeate under reduced pressure and freeze dry to obtain the flaxseed meal oligosaccharide extract; wherein, the amount of enzyme added is 2-5% of the mass of the raw material extracted in step S1;

[0054] The enzyme is selected from cellulase, pectinase or xylanase; the freeze-drying conditions are: vacuum degree 10-50 Pa, temperature -20 to 40℃, and duration 24-48 h.

[0055] The above method was used to prepare flaxseed meal oligosaccharide extract with blood sugar regulation, with a yield (based on total flaxseed meal) of 4.65-5.05% and an oligosaccharide content of over 92%.

[0056] A method for preparing an extract of fraxinus erythrolene ethers from fraxinus includes the following steps:

[0057] (1) The crushed and sieved Qinpi was dispersed in DES aqueous solution at a material-to-liquid ratio of 1:(20-40)g / mL to obtain a mixed solution;

[0058] The DES aqueous solution is prepared by mixing DES solvent and water, wherein the mass percentage of water in the DES aqueous solution is 10-30%, and the DES solvent is prepared by mixing choline chloride and oxalic acid in a molar ratio of 1:2-3, or the DES solvent is prepared by mixing choline chloride and polyol in a molar ratio of 1:2-3, wherein the polyol is selected from 1,4-butanediol or glycerol.

[0059] (2) Add cellulase to the mixture obtained in step (1), mix well, microwave extract at 40-60℃ for 30-60 min, filter, take the filtrate, concentrate under reduced pressure to a mass-volume fraction of 0.3-0.5 kg / L, centrifuge and separate the supernatant;

[0060] The amount of cellulase added is 2-5% of the mass of the Fraxinus chinensis bark in step (1); the microwave extraction is performed with a microwave power of 450-550W; the centrifugation speed is controlled at 4500-5500rpm and the duration is 8-12min.

[0061] (3) Inject the supernatant obtained in step (2) into the adsorption resin, first wash with water until the eluent is nearly colorless, discard the eluent, then wash with 20-40 vol% ethanol aqueous solution to remove impurities, discard the eluent, and finally wash with 80-90 vol% ethanol aqueous solution. Collect the eluent of 80-90 vol% ethanol aqueous solution, concentrate under reduced pressure, and freeze dry to obtain the product.

[0062] The adsorption resin used is a macroporous resin of type AB-8, D101, or NKA-9, and the amount of adsorption resin used is 1-1.5 times the mass of the Fraxinus bark. When eluting with water, the amount of water used is 5-8 times the mass of the Fraxinus bark, and the elution rate is 1.0-2.0 Bv / h. When eluting with 20-40 vol% ethanol aqueous solution, the amount of 20-40 vol% ethanol aqueous solution used is 5-8 times the mass of the Fraxinus bark, and the elution rate is 1.0-2.0 Bv / h. When eluting with 80-90 vol% ethanol aqueous solution, the amount of 80-90 vol% ethanol aqueous solution used is 9-12 times the mass of the Fraxinus bark, and the elution rate is 1.0-2.0 Bv / h. The freeze-drying conditions are: 10-50 Pa, temperature -20 to 40℃, and duration 24-48 h.

[0063] The above method was used to prepare an extract of cyclohexene ethers from Fraxinus rhynchophylla, with a yield (based on total Fraxinus rhynchophylla amount) of 3.20-3.83% and a content of cyclohexene ethers in the extract of Fraxinus rhynchophylla of over 82%.

[0064] A hypoglycemic composition comprising the following raw materials in parts by weight: 4-9 parts of flaxseed meal oligosaccharide extract and 3-6 parts of fraxinus rhizome iridoid extract; this hypoglycemic composition may be used in hypoglycemic foods, medicines or health products.

[0065] Example 1

[0066] A method for preparing a flaxseed meal oligosaccharide extract with blood sugar regulating properties includes the following steps:

[0067] S1. Thoroughly dry and pulverize flaxseed meal, pass it through a 40-mesh sieve, and sterilize it (sterilize at 0.1 MPa and 121℃ for 20 min) as the extraction raw material. Mix the extraction raw material with an aqueous solution containing fermentation bacteria at a mass ratio of 1:5, and ferment at 28℃ for 48 h to obtain the fermentation product; wherein, the volume fraction of the fermentation bacteria suspension in the aqueous solution containing fermentation bacteria is 3% (the concentration of fermentation bacteria in the aqueous solution containing fermentation bacteria is 3×10⁻⁶). 7 (CFU / mL)

[0068] S2. Add water equal to 8 times the weight of the raw material to the fermentation product obtained in step S1, and extract at 85℃ with stirring for 1.5h. Then filter, take the filtrate, concentrate under reduced pressure to a mass-volume fraction of 0.3kg / L, stop the concentration, centrifuge (5000rpm, 10min) and separate the supernatant.

[0069] S3. Add 95 vol% ethanol aqueous solution to the product obtained in step S2, mix well to obtain a mixed solution containing 80 vol% ethanol, let the mixed solution stand at 4℃ for 24 h, centrifuge (5000 rpm, 10 min) to obtain a precipitate, add 1 times the weight of water to the precipitate to dissolve it, and obtain an aqueous solution of the extract.

[0070] S4. Add cellulase to the aqueous extract obtained in step S3, heat to 40℃ for 3 hours of enzymatic hydrolysis, then inactivate the enzyme in a boiling water bath for 5 minutes, filter, and collect the filtrate to obtain the enzymatic hydrolysate; place the enzymatic hydrolysate in a dialysis bag with a molecular weight of 300 Da, change the water 3 times at 8h, 12h and 16h respectively, and dialyze for a total of 24h, collect the retaining liquid; then place the retaining liquid in a dialysis bag with a molecular weight of 2000 Da, dialyze for 24h, collect the permeate, concentrate under reduced pressure and freeze dry to obtain the flaxseed meal oligosaccharide extract; wherein, the amount of cellulase added is 2% of the mass of the raw material; the freeze drying conditions are: vacuum degree 50Pa, temperature -30℃, duration 24h.

[0071] The above method was used to prepare flaxseed meal oligosaccharide extract with blood sugar regulation, with a yield (based on total flaxseed meal) of 4.65% and an oligosaccharide content of 92.54%.

[0072] Example 2

[0073] A method for preparing a flaxseed meal oligosaccharide extract with blood sugar regulating properties includes the following steps:

[0074] S1. Thoroughly dry and pulverize flaxseed meal, pass it through a 40-mesh sieve, and sterilize it (sterilize at 0.1 MPa and 121℃ for 20 min). Use this as the extraction raw material. Mix the extraction raw material with an aqueous solution containing fermentation bacteria at a mass ratio of 1:6, and ferment at 30℃ for 36 h to obtain the fermentation product. The volume fraction of the fermentation bacteria suspension in the aqueous solution containing fermentation bacteria is 5% (i.e., the concentration of fermentation bacteria in the aqueous solution containing fermentation bacteria is 5 × 10⁻⁶). 7 (CFU / mL)

[0075] S2. Add 10 times the weight of water to the fermentation product obtained in step S1 to dilute the raw material, and extract at 90℃ with stirring for 2.0h. Then filter, take the filtrate, concentrate under reduced pressure to a mass-volume fraction of 0.4kg / L, stop the concentration, centrifuge (5000rpm, 10min) and separate the supernatant.

[0076] S3. Add 95 vol% ethanol aqueous solution to the product obtained in step S2, mix well to obtain a mixed solution containing 80 vol% ethanol, let the mixed solution stand at 4℃ for 24 h, centrifuge (5000 rpm, 10 min) to obtain a precipitate, add 1.2 times the weight of water to the precipitate to dissolve it, and obtain an aqueous solution of the extract.

[0077] S4. Add pectinase to the aqueous extract obtained in step S3, heat to 50℃ for 4 hours of enzymatic hydrolysis, then inactivate the enzyme in a boiling water bath for 5 minutes, filter, and collect the filtrate to obtain the enzymatic hydrolysis extract; place the enzymatic hydrolysis extract in a dialysis bag with a molecular weight of 300 Da, change the water 3 times at 8h, 12h and 16h respectively, and dialyze for a total of 24h, collect the retaining liquid; then place the retaining liquid in a dialysis bag with a molecular weight of 2000 Da, dialyze for 24h, collect the permeate, concentrate under reduced pressure and freeze dry to obtain the flaxseed meal oligosaccharide extract; wherein, the amount of pectinase added is 3% of the mass of the raw material; the freeze drying conditions are: vacuum degree 20Pa, temperature -40℃, duration 36h.

[0078] The above method was used to prepare an oligosaccharide extract of flaxseed meal that regulates blood sugar, with a yield (based on the total amount of flaxseed meal) of 4.81% and an oligosaccharide content of 92.76%.

[0079] Example 3

[0080] A method for preparing a flaxseed meal oligosaccharide extract with blood sugar regulating properties includes the following steps:

[0081] S1. Thoroughly dry and pulverize flaxseed meal, pass it through a 40-mesh sieve, and sterilize it (sterilize at 0.1 MPa and 121℃ for 20 min) as the extraction raw material. Mix the extraction raw material with an aqueous solution containing fermentation bacteria at a mass ratio of 1:7, and ferment at 32℃ for 72 h to obtain the fermentation product; wherein, the volume fraction of the fermentation bacteria suspension in the aqueous solution containing fermentation bacteria is 3% (i.e., the concentration of fermentation bacteria in the aqueous solution containing fermentation bacteria is 3×10⁻⁶). 7 (CFU / mL)

[0082] S2. Add 12 times the weight of water to the fermentation product obtained in step S1 to dilute the raw material, stir and extract at 100℃ for 3 hours, then filter, take the filtrate, concentrate under reduced pressure to a mass-volume fraction of 0.5 kg / L, stop the concentration, centrifuge (5000 rpm, 10 min) and separate the supernatant.

[0083] S3. Add 95 vol% ethanol aqueous solution to the product obtained in step S2, mix well to obtain a mixed solution containing 80 vol% ethanol, let the mixed solution stand at 4℃ for 24 h, centrifuge (5000 rpm, 10 min) to obtain a precipitate, add 1.5 times the weight of water to the precipitate to dissolve it, and obtain an aqueous solution of the extract.

[0084] S4. Add xylanase to the aqueous extract obtained in step S3, heat to 60℃ for 5 hours of enzymatic hydrolysis, then inactivate the enzyme in a boiling water bath for 5 minutes, filter, and collect the filtrate to obtain the enzymatic hydrolysis extract; place the enzymatic hydrolysis extract in a dialysis bag with a molecular weight of 300 Da, change the water three times at 8h, 12h and 16h respectively, and dialyze for a total of 24 hours, and collect the retention liquid; then place the retention liquid in a dialysis bag with a molecular weight of 2000 Da, dialyze for 24 hours, collect the permeate, concentrate under reduced pressure and freeze dry to obtain the flaxseed meal oligosaccharide extract; wherein, the amount of xylanase added is 5% of the mass of the raw material; the freeze drying conditions are: vacuum degree 10 Pa, temperature -30℃, duration 36 hours.

[0085] The above method was used to prepare an oligosaccharide extract of flaxseed meal that regulates blood sugar, with a yield (based on the total amount of flaxseed meal) of 5.05% and an oligosaccharide content of 93.34%.

[0086] Example 4

[0087] A method for preparing an extract of fraxinus erythrolene ethers from fraxinus includes the following steps:

[0088] (1) The crushed and sieved Qinpi (Qinpi raw material) is dispersed in DES aqueous solution at a material-to-liquid ratio of 1:20 g / mL to obtain a mixed solution; wherein the DES aqueous solution is formed by mixing DES solvent and water, the mass percentage of water in the DES aqueous solution is 10%, and the DES solvent is formed by mixing choline chloride and 1,4-butanediol at a molar ratio of 1:2.

[0089] (2) Add cellulase to the mixture obtained in step (1), mix well, extract by microwave (500W) at 40℃ for 30 min, filter, take the filtrate, concentrate under reduced pressure to a mass-volume fraction of 0.35 kg / L, centrifuge (5000 rpm, 10 min), and separate the supernatant; wherein, the amount of cellulase added is 2% of the mass of the Fraxinus chinensis raw material;

[0090] (3) Inject the supernatant obtained in step (2) into the adsorption resin, first wash with water until the eluent is nearly colorless, discard the eluent, then wash with 20 vol% ethanol aqueous solution to remove impurities, discard the eluent, and finally wash with 80 vol% ethanol aqueous solution. Collect the eluent of 80 vol% ethanol aqueous solution, concentrate under reduced pressure, and freeze dry to obtain the product.

[0091] The adsorption resin used is AB-8 macroporous resin, and the amount of adsorption resin is 1 times the mass of the Fraxinus bark. When eluting with water, the amount of water is 6 times the mass of the Fraxinus bark, and the elution rate is 1.2 Bv / h. When eluting with 20 vol% ethanol aqueous solution, the amount of 20 vol% ethanol aqueous solution is 5 times the mass of the Fraxinus bark, and the elution rate is 1.5 Bv / h. When eluting with 80 vol% ethanol aqueous solution, the amount of 80 vol% ethanol aqueous solution is 10 times the mass of the Fraxinus bark, and the elution rate is 1.5 Bv / h. The freeze-drying conditions are: vacuum degree 30 Pa, temperature -40℃, and duration 24 h.

[0092] The cyclohexene ether terpene extract of Fraxinus rhynchophylla was prepared by the above method, with a yield (based on total Fraxinus rhynchophylla amount) of 3.20% and a content of cyclohexene ether terpene components of 82.14%.

[0093] Example 5

[0094] A method for preparing an extract of fraxinus erythrolene ethers from fraxinus includes the following steps:

[0095] (1) The crushed and sieved Qinpi (Qinpi raw material) is dispersed in DES aqueous solution at a material-to-liquid ratio of 1:30 g / mL to obtain a mixed solution; wherein the DES aqueous solution is formed by mixing DES solvent and water, the mass percentage of water in the DES aqueous solution is 20%, and the DES solvent is formed by mixing choline chloride and oxalic acid at a molar ratio of 1:2.

[0096] (2) Add cellulase to the mixture obtained in step (1), mix well, extract by microwave (500W) at 50℃ for 45 min, filter, take the filtrate, concentrate under reduced pressure to a mass-volume fraction of 0.35 kg / L, centrifuge (5000 rpm, 10 min), and separate the supernatant; wherein, the amount of cellulase added is 4% of the mass of the Fraxinus bark raw material;

[0097] (3) Inject the supernatant obtained in step (2) into the adsorption resin, first wash with water until the eluent is nearly colorless, discard the eluent, then wash with 30 vol% ethanol aqueous solution to remove impurities, discard the eluent, and finally wash with 80 vol% ethanol aqueous solution. Collect the eluent of 80 vol% ethanol aqueous solution, concentrate under reduced pressure, and freeze dry to obtain the product.

[0098] The adsorption resin used is a macroporous resin of type D101, and the amount of adsorption resin is 1.2 times the mass of the Fraxinus bark. When eluting with water, the amount of water is 5 times the mass of the Fraxinus bark, and the elution rate is 1.0 Bv / h. When eluting with 30 vol% ethanol aqueous solution, the amount of 30 vol% ethanol aqueous solution is 8 times the mass of the Fraxinus bark, and the elution rate is 1.5 Bv / h. When eluting with 80 vol% ethanol aqueous solution, the amount of 80 vol% ethanol aqueous solution is 11 times the mass of the Fraxinus bark, and the elution rate is 1.2 Bv / h. The freeze-drying conditions are: vacuum degree 15 Pa, temperature -35℃, and duration 24 h.

[0099] The extract of iridoid ethers from Fraxinus rhynchophylla was prepared using the above method, with a yield (based on total Fraxinus rhynchophylla amount) of 3.44% and an iridoid ether content of 83.45% in the extract.

[0100] Example 6

[0101] A method for preparing an extract of fraxinus erythrolene ethers from fraxinus includes the following steps:

[0102] (1) The crushed and sieved Qinpi (Qinpi raw material) is dispersed in DES aqueous solution at a material-to-liquid ratio of 1:35 g / mL to obtain a mixed solution; wherein the DES aqueous solution is formed by mixing DES solvent and water, the mass percentage of water in the DES aqueous solution is 10%, and the DES solvent is formed by mixing choline chloride and glycerol at a molar ratio of 1:3.

[0103] (2) Add cellulase to the mixture obtained in step (1), mix well, extract by microwave (500W) at 60℃ for 60 min, filter, take the filtrate, concentrate under reduced pressure to a mass-volume fraction of 0.5 kg / L, centrifuge (5000 rpm, 10 min), and separate the supernatant; wherein, the amount of cellulase added is 5% of the mass of the Fraxinus chinensis raw material;

[0104] (3) Inject the supernatant obtained in step (2) into the adsorption resin, first wash with water until the eluent is nearly colorless, discard the eluent, then wash with 20 vol% ethanol aqueous solution to remove impurities, discard the eluent, and finally wash with 90 vol% ethanol aqueous solution. Collect the eluent of 90 vol% ethanol aqueous solution, concentrate under reduced pressure, and freeze dry to obtain the product.

[0105] The adsorption resin used is a macroporous resin of type NKA-9, and the amount of adsorption resin is 1.5 times the mass of the Fraxinus bark. When eluting with water, the amount of water is 8 times the mass of the Fraxinus bark, and the elution rate is 1.8 Bv / h. When eluting with 20 vol% ethanol aqueous solution, the amount of 20 vol% ethanol aqueous solution is 6 times the mass of the Fraxinus bark, and the elution rate is 1.5 Bv / h. When eluting with 90 vol% ethanol aqueous solution, the amount of 90 vol% ethanol aqueous solution is 12 times the mass of the Fraxinus bark, and the elution rate is 1.2 Bv / h. The freeze-drying conditions are: vacuum degree 10 Pa, temperature -40℃, and duration 36 h.

[0106] The extract of iridoid ethers from Fraxinus rhynchophylla was prepared using the above method, with a yield (based on total Fraxinus rhynchophylla amount) of 3.83% and an iridoid ether content of 82.56% in the extract.

[0107] Example 7

[0108] A hypoglycemic composition comprising the following raw materials in parts by weight: 5 parts of flaxseed meal oligosaccharide extract as described in Example 1, and 3 parts of fraxinus rhizome ether terpene extract as described in Example 4.

[0109] Example 8

[0110] A hypoglycemic composition comprising the following raw materials in parts by weight: 7 parts of flaxseed meal oligosaccharide extract as described in Example 1, and 4 parts of fraxinus rhizome ether terpene extract as described in Example 4.

[0111] Example 9

[0112] A hypoglycemic composition comprising the following raw materials in parts by weight: 9 parts of flaxseed meal oligosaccharide extract as described in Example 1, and 6 parts of fraxinus rhizome ether terpene extract as described in Example 4.

[0113] Comparative Example 1

[0114] A method for preparing an oligosaccharide extract from flaxseed meal includes the following steps:

[0115] S1. Thoroughly dry and pulverize the flaxseed meal, pass it through a 40-mesh sieve, and sterilize it (sterilize it at 0.1MPa and 121℃ for 20 minutes) as the extraction raw material. Mix the extraction raw material with water at a mass ratio of 1:5 and soak it at 28℃ for 48 hours to obtain the soaking product.

[0116] S2. Add water equal to 8 times the weight of the raw material to the soaking product obtained in step S1, and extract at 85℃ with stirring for 1.5h. Then filter, take the filtrate, concentrate under reduced pressure to a mass-volume fraction of 0.3kg / L, stop the concentration, centrifuge (5000rpm, 10min) and separate the supernatant.

[0117] S3. Add 95 vol% ethanol aqueous solution to the product obtained in step S2, mix well to obtain a mixed solution containing 80 vol% ethanol, let the mixed solution stand at 4℃ for 24 h, centrifuge (5000 rpm, 10 min) to obtain a precipitate, add 1 times the weight of water to the precipitate to dissolve it, and obtain an aqueous solution of the extract.

[0118] S4. Add cellulase to the aqueous extract obtained in step S3, heat to 40℃ for 3 hours of enzymatic hydrolysis, then inactivate the enzyme in a boiling water bath for 5 minutes, filter, and collect the filtrate to obtain the enzymatic extract; place the enzymatic extract in a dialysis bag with a molecular weight of 300 Da, change the water 3 times at 8h, 12h and 16h respectively, and dialyze for a total of 24h, and collect the retention liquid; then place the retention liquid in a dialysis bag with a molecular weight of 2000 Da, dialyze for 24h, collect the permeate, concentrate under reduced pressure and freeze dry to obtain the flaxseed meal oligosaccharide extract; wherein, the amount of enzyme added is 2% of the mass of the raw material; the freeze drying conditions are: vacuum degree 50Pa, temperature -30℃, duration 24h.

[0119] The preparation method of Comparative Example 1 differs from that of Example 1 in that *Trichoderma hygroscopicum* was not added in step S1 of Comparative Example 1. The flaxseed meal oligosaccharide extract prepared using the method described in Comparative Example 1 had a yield (based on the total amount of flaxseed meal) of 1.06%, and an oligosaccharide content of 90.54%.

[0120] Comparative Example 2

[0121] A method for preparing an oligosaccharide extract from flaxseed meal includes the following steps:

[0122] S1. Thoroughly dry and pulverize flaxseed meal, pass it through a 40-mesh sieve, and sterilize it (sterilize at 0.1 MPa and 121℃ for 20 min) to obtain the extraction raw material. Mix the extraction raw material with water at a mass ratio of 1:5 to obtain a dispersion. Add cellulase to the dispersion and heat to 40℃ for enzymatic hydrolysis for 3 h. The amount of cellulase added is 2% of the mass of the extraction raw material.

[0123] S2. Add water equal to 8 times the weight of the raw material to the product obtained in step S1, and extract at 85℃ with stirring for 1.5h. Then filter, take the filtrate, concentrate under reduced pressure to a mass-volume fraction of 0.3kg / L, stop the concentration, centrifuge (5000rpm, 10min) and separate the supernatant.

[0124] S3. Add 95 vol% ethanol aqueous solution to the product obtained in step S2, mix well to obtain a mixed solution containing 80 vol% ethanol, let the mixed solution stand at 4℃ for 24 h, centrifuge (5000 rpm, 10 min) to obtain a precipitate, add 1 times the weight of water to the precipitate to dissolve it, and obtain an aqueous solution of the extract.

[0125] S4. Add cellulase to the aqueous extract obtained in step S3, heat to 40℃ for 3 hours of enzymatic hydrolysis, then inactivate the enzyme in a boiling water bath for 5 minutes, filter, and collect the filtrate to obtain the enzymatic extract; place the enzymatic extract in a dialysis bag with a molecular weight of 300 Da, change the water 3 times at 8h, 12h and 16h respectively, and dialyze for a total of 24h, and collect the retention liquid; then place the retention liquid in a dialysis bag with a molecular weight of 2000 Da, dialyze for 24h, collect the permeate, concentrate under reduced pressure and freeze dry to obtain the flaxseed meal oligosaccharide extract; wherein, the amount of enzyme added is 2% of the mass of the aqueous extract; the freeze drying conditions are: vacuum degree 50Pa, temperature -30℃, duration 24h.

[0126] The preparation method of Comparative Example 2 differs from that of Example 1 in that cellulase was added instead of *Streptomyces cuspidata* in step S1 of Comparative Example 2. The flaxseed meal oligosaccharide extract prepared using the method described in Comparative Example 2 had a yield (based on total flaxseed meal) of 0.97% and an oligosaccharide content of 92.31%.

[0127] Comparative Example 3

[0128] A method for preparing an oligosaccharide extract from flaxseed meal includes the following steps:

[0129] S1. Thoroughly dry and pulverize the flaxseed meal, pass it through a 40-mesh sieve, and sterilize it (sterilize it at 0.1MPa and 121℃ for 20 minutes) to use as the extraction raw material. Add 8 times the weight of water to the extraction raw material to dilute it, and extract it at a constant temperature of 85℃ for 1.5 hours. Then filter and collect the filtrate.

[0130] S2. Add cellulase to the filtrate obtained in step S1, heat to 40℃ for 3 hours for enzymatic hydrolysis, then inactivate the enzyme in a boiling water bath for 5 minutes, filter, and collect the filtrate. The amount of cellulase added is 2% of the mass of the filtrate obtained in step S1.

[0131] S3. Add 95 vol% ethanol aqueous solution to the product obtained in step S2, mix well to obtain a mixed solution containing 80 vol% ethanol, let the mixed solution stand at 4℃ for 24 h, centrifuge (5000 rpm, 10 min) to obtain a precipitate, add 1 times the weight of water to the precipitate to dissolve it, and obtain an aqueous solution of the extract.

[0132] S4. Add *Saccharomyces cerevisiae* (3% of the extract mass) to the aqueous extract obtained in step S3, and ferment at 28°C for 48 hours to obtain the fermentation product. Filter the product, collect the filtrate, sterilize it, and obtain the fermentation extract. Place the fermentation extract in a dialysis bag with a molecular weight of 300 Da, and change the water three times at 8, 12, and 16 hours of dialysis, for a total of 24 hours. Collect the retained solution. Then place the retained solution in a dialysis bag with a molecular weight of 2000 Da, dialyze for 24 hours, collect the permeate, concentrate under reduced pressure, and freeze-dry (vacuum degree 50 Pa, temperature -30°C, duration 24 hours) to obtain the flaxseed meal oligosaccharide extract.

[0133] The preparation method of Comparative Example 3 differs from that of Example 1 in that Comparative Example 4 follows the method described in the background art (CN201910098976.X), first involving extraction with heated water, followed by enzymatic hydrolysis, alcohol precipitation, and fermentation. The flaxseed meal oligosaccharide extract prepared using the method described in Comparative Example 4 had a yield (based on the total amount of flaxseed meal) of 1.21%, and an oligosaccharide content of 90.61%.

[0134] A comparison of Comparative Examples 1-3 with Example 1 shows that each step in fermentation, extraction, alcohol precipitation, and enzymatic hydrolysis affects the yield of the target product. Omitting fermentation or postponing the fermentation step, or even adding an enzymatic hydrolysis step, will significantly reduce the yield. The yield of the present invention is more than 3.84 times that of the comparative examples.

[0135] Comparative Example 4

[0136] A method for preparing an extract of fraxinus erythrolene ethers from fraxinus includes the following steps:

[0137] (1) Disperse the crushed and sieved Qinpi in water at a material-to-liquid ratio of 1:20 g / mL to obtain a mixed liquid;

[0138] (2) Extract the mixture obtained in step (1) at 40℃ using microwave (500W) for 30 min, filter, take the filtrate, concentrate under reduced pressure to a mass volume fraction of 0.35 kg / L, centrifuge (5000 rpm, 10 min), and separate the supernatant;

[0139] (3) Inject the supernatant obtained in step (2) into the adsorption resin, first wash with water until the eluent is nearly colorless, discard the eluent, then wash with 20 vol% ethanol aqueous solution to remove impurities, discard the eluent, and finally wash with 80 vol% ethanol aqueous solution. Collect the eluent of 80 vol% ethanol aqueous solution, concentrate under reduced pressure, and freeze dry to obtain the product.

[0140] The adsorption resin used is AB-8 macroporous resin, and the amount of adsorption resin is 1.5 times the mass of the Fraxinus bark. When eluting with water, the amount of water is 5 times the mass of the Fraxinus bark, and the elution rate is 1.2 Bv / h. When eluting with 20% ethanol aqueous solution, the amount of 20% ethanol aqueous solution is 6 times the mass of the Fraxinus bark, and the elution rate is 1.2 Bv / h. When eluting with 80% ethanol aqueous solution, the amount of 80% ethanol aqueous solution is 10 times the mass of the Fraxinus bark, and the elution rate is 1.2 Bv / h. The freeze-drying conditions are: vacuum degree 20 Pa, temperature -30℃, and duration 24 h.

[0141] The extract of cyclohexene ethers from Fraxinus rhynchophylla was prepared using the above method, with a yield (based on total Fraxinus rhynchophylla) of 2.13% and a content of cyclohexene ethers in the extract of Fraxinus rhynchophylla of 52.13%. Compared with Example 4, Comparative Example 4 differs in that DES solvent (eutectic solvent) was not used in step (1) of Comparative Example 5, and cellulase was not used in step (2). The yield and content of Comparative Example 4 were significantly reduced.

[0142] Application Trial

[0143] Experiment 1: Evaluation of the in vitro hypoglycemic activity of the composition

[0144] Alpha-glucosidase and α-amylase play crucial roles in the metabolism of dietary carbohydrates, promoting the breakdown of starch, sucrose, maltose, and other substances into monosaccharides. Excessive enzyme activity can lead to a sharp rise in postprandial blood glucose. Currently, one of the commonly used methods for controlling blood glucose in diabetic patients is to inhibit the activity of α-glucosidase and α-amylase, reducing the rate at which starches are broken down into glucose in the body, thereby lowering postprandial blood glucose. Therefore, studies on the inhibitory effects of α-glucosidase and α-amylase are commonly used research methods for evaluating their in vitro hypoglycemic activity.

[0145] This experiment used the flaxseed meal oligosaccharide extract described in Example 2, the fraxinus chinensis iridoid extract described in Example 5, and the hypoglycemic compositions described in Examples 7-9 as test samples to detect their inhibitory activities on α-glucosidase and α-amylase, and to investigate the in vitro hypoglycemic effects of the flaxseed meal oligosaccharide extract, the fraxinus chinensis iridoid extract, and the hypoglycemic compositions.

[0146] (1) Assay of α-glucosidase inhibitory activity

[0147] Prepare a phosphate-buffered saline (PBS) solution at pH 6.8, using acarbose as a positive control. Add 20 μL of sample solutions of different concentrations to a 96-well plate, then add 20 μL of α-glucosidase solution (0.4 U / mL), mix thoroughly, and incubate at a pre-stabilized 37°C for 10 min. Remove the plate, add 20 μL of pNPG solution (2.5 mmol / L), mix thoroughly, and incubate at 37°C for 30 min. Finally, add 80 μL of sodium carbonate solution (0.5 mol / L), and measure the absorbance (A value) at 405 nm. For the blank background group, use PBS solution instead of the sample solutions and α-glucosidase solution.

[0148] The α-glucosidase inhibition rate was calculated using the following formula, and the results are as follows: Figure 1 As shown.

[0149] α-glucosidase inhibition rate (%) =

[0150] In the formula: A1 represents the absorbance of the experimental sample (sample and α-glucosidase); A2 represents the absorbance of the sample background group (sample without α-glucosidase); A3 represents the absorbance of the blank group (PBS and α-glucosidase); A4 represents the absorbance of the blank background group (PBS).

[0151] Depend on Figure 1 It is evident that the flaxseed meal oligosaccharide extract described in Example 2, the Fraxinus chinensis iridoid extract described in Example 5, and the hypoglycemic compositions described in Examples 7-9 all exhibit a certain inhibitory effect on α-glucosidase, and the inhibition rate of α-glucosidase increases with increasing concentration. Among these, the hypoglycemic composition shows a significantly better inhibitory effect on α-glucosidase than the flaxseed meal oligosaccharide extract and the Fraxinus chinensis iridoid extract, and when the mass concentration is greater than 0.64 mg / mL, the inhibition rate reaches over 90%, which is essentially equivalent to the inhibition rate of acarbose. This indicates that the flaxseed meal oligosaccharide extract and the Fraxinus chinensis iridoid extract prepared in this invention have a synergistic effect, and the hypoglycemic composition can effectively inhibit α-glucosidase activity, exhibiting good in vitro hypoglycemic activity.

[0152] (2) Assay of α-amylase inhibitory activity

[0153] Prepare a phosphate-buffered saline (PBS) solution at pH 6.8, using acarbose as a positive control. Take 250 μL of sample solutions of different concentrations into centrifuge tubes, add 250 μL of α-amylase solution (1.68 U / mL), mix well, and incubate at a pre-stabilized 37°C for 15 min. Remove from the incubator, add 250 μL of soluble starch solution (1%), mix well, and incubate at 37°C for 15 min. Finally, add 1 mL of DNS solution and incubate in a boiling water bath for 15 min to terminate the reaction. Measure the absorbance (A value) at 591 nm. For the blank background group, use PBS solution instead of the sample solution and α-amylase solution.

[0154] The α-amylase inhibition rate was calculated using the following formula, and the results are as follows: Figure 2 As shown.

[0155] α-Amylase inhibition rate (%) =

[0156] In the formula: A1 represents the absorbance of the experimental sample (sample and α-amylase solution); A2 represents the absorbance of the sample background group (sample without α-amylase solution); A3 represents the absorbance of the blank group (PBS and α-amylase solution); A4 represents the absorbance of the blank background group (PBS).

[0157] Depend on Figure 2 It can be seen that the flaxseed meal oligosaccharide extract described in Example 2, the Fraxinus chinensis iridoid extract described in Example 5, and the hypoglycemic compositions described in Examples 7-9 all have a certain inhibitory effect on α-amylase, and the inhibition rate of α-amylase increases with increasing concentration. Among them, the hypoglycemic composition has a significantly better inhibitory effect on α-amylase than the flaxseed meal oligosaccharide extract and the Fraxinus chinensis iridoid extract. This indicates that the flaxseed meal oligosaccharide extract and the Fraxinus chinensis iridoid extract prepared in this invention have a synergistic effect, and the hypoglycemic composition can effectively inhibit α-amylase activity, exhibiting good in vitro hypoglycemic activity.

[0158] Experiment 2: Effects of the combined composition on insulin resistance in HepG2 cells

[0159] Diabetes mellitus is a metabolic disease characterized by hyperglycemia, with type 2 diabetes mellitus (T2DM), characterized by insulin resistance, accounting for more than 90% of diabetes cases. The development and progression of type 2 diabetes are closely related to impaired insulin signaling and decreased insulin sensitivity caused by insulin resistance (IR). Insulin regulates blood glucose homeostasis by mediating its biological effects through insulin receptors in target cells. As the primary target organ for insulin action, the liver, muscles, and adipose tissue experience impaired glucose uptake and increased glucose output in cases of insulin resistance, leading to impaired glucose homeostasis and elevated blood glucose. Given the prevalence of liver damage among diabetes-related complications, addressing hepatic insulin insensitivity holds promise as a potential treatment for diabetes.

[0160] This experiment used a HepG2 hepatocyte insulin resistance model induced by simultaneous high glucose and insulin. The flaxseed meal oligosaccharide extract described in Example 2, the Fraxinus chinensis iridoid extract described in Example 5, and the hypoglycemic compositions described in Examples 7-9 were used as test samples to detect their effects on HepG2 hepatocyte insulin resistance model cell viability, glucose consumption in the culture medium, hexokinase (HK) and pyruvate kinase (PK) activity in the cells, and glycogen content. The hypoglycemic activities and mechanisms of the flaxseed meal oligosaccharide extract, the Fraxinus chinensis iridoid extract, and the compositions of this invention were investigated.

[0161] (1) Cell culture

[0162] After resuscitation, HepG2 liver cancer cells were added to DMEM complete medium (5.5 mM glucose, a mixture of 10% fetal bovine serum and 1% penicillin / streptomycin) and cultured in an incubator (37°C, 5% CO2). Cells were passaged when the cell density reached approximately 80%. Cells in the logarithmic growth phase were selected for experiments.

[0163] (2) Cell viability was determined by the CCK-8 assay.

[0164] The experiment consisted of an experimental group, a control group, and a blank group. The experimental group received different concentrations of flaxseed meal oligosaccharide extract, fraxinus erythrolene ether extract, and an antihypertensive composition (500.00, 250.00, 125.00, 62.50, 31.25, 15.63, and 7.81 μg / mL). The control group consisted of normally cultured cells without any drugs. The blank group contained culture medium without cells or any drugs. Cell pellets were collected and diluted to a density of 1 × 10⁻⁶ cells / mL. 5Cells / mL were collected, and 100 μL of cell suspension was added to each well of a 96-well plate. After culturing for 24 h, 100 μL of culture medium containing different concentrations of the sample was added to each well for the experimental group, and 100 μL of complete culture medium was added to each blank group and control group. Each group was repeated 6 times for 24 h of pretreatment. Then, 100 μL of culture medium containing 10% CCK-8 was added by changing the medium, and the plates were incubated at 37°C for 2 h. The OD value at 450 nm was measured using a microplate reader. With normal cells as the control, the cell viability was calculated using the following formula:

[0165] Cell viability (%) = [(OD) 试验 -OD 空白 ) / (OD 对照 -OD 空白 )]×100%

[0166] The results are shown in Table 1.

[0167] Table 1. Effects of the compositions of the present invention on the activity of HepG2 cells (unit: %)

[0168]

[0169] As shown in Table 1, the flaxseed meal oligosaccharide extract prepared in Example 2, the fraxinus erythrolene ether extract prepared in Example 5, and the hypoglycemic compositions prepared in Examples 7-9, at concentrations of 500 μg / mL or lower, did not affect the proliferation of HepG2 cells, and cell viability was above 90%, indicating that each sample was essentially non-cytotoxic. Therefore, a sample concentration of 500 μg / mL was selected for the next step of the experiment.

[0170] (3) Effect on glucose consumption in insulin-resistant (IR) HepG2 cells

[0171] The experiment consisted of an experimental group, a positive control group, a model group, a normal group, and a blank group. The experimental group contained 500 μg / mL of the flaxseed meal oligosaccharide extract described in Example 2, the fraxinus rhizome extract described in Example 5, and the hypoglycemic compositions described in Examples 7-9. Cell pellets were collected and adjusted to 1×10⁻⁶ DMEM complete medium. 5Cell suspensions of 100 µL / mL were seeded into 96-well plates and cultured for 24 h, after which the culture medium was discarded. The experimental group, positive control group, and model group were treated with 100 µL of serum-free DMEM medium (25 mM glucose) containing 10 nM insulin. The normal group was treated with 100 µL of serum-free DMEM medium (5.5 mM glucose), and the blank group with 100 µL of cell-free serum-free DMEM medium (5.5 mM glucose). Each group was repeated in 6 wells. After culturing for another 36 h, the culture medium was discarded. The experimental group was treated with sample culture medium containing 500 μg / mL, the positive control group with metformin culture medium containing 200 μg / mL, and the model and normal groups with the same amount of complete culture medium. After 24 h, the glucose content in the culture medium was measured according to the glucose assay kit instructions. The glucose consumption for each group was calculated as follows: Glucose consumption (mmol / L) = Glucose content in cell-free medium - Glucose content in cell-containing medium. The results are shown below. Figure 3 As shown in the figure (different letters indicate significant differences), P <0.05).

[0172] like Figure 3 As shown, compared with the normal group, the glucose consumption in the model group was significantly reduced ( P <0.05 indicates abnormal glucose metabolism and insulin resistance in the cells, confirming successful modeling. Treatment of IR-HepG2 cells with flaxseed meal oligosaccharide extract, fraxinus erythropoietin extract, and a hypoglycemic combination significantly increased glucose consumption. P <0.05%, among which the hypoglycemic composition, after acting on IR-HepG2 cells, showed significantly better glucose consumption than the extracts of flaxseed meal oligosaccharides and fraxinus ethers. P The concentration of insulin (<0.05) is almost comparable to that of metformin at 200 μg / mL in improving insulin resistance. This indicates that the hypoglycemic composition can effectively alleviate insulin resistance in HepG2 cells induced by high glucose and insulin by significantly increasing glucose consumption, demonstrating a good hypoglycemic effect.

[0173] (5) Effects on glucose metabolism pathways in IR-HepG2 cells

[0174] Collect the cell pellet and add DMEM complete medium to adjust to 5 × 10⁻⁶. 4Cell suspension of 1 cell / mL was seeded into 6-well plates at 2 mL / well and cultured for 24 h. Experimental group, positive control group, model group, normal group, and blank group were set up, with the remaining procedures the same as the previous experiment. After the sample treatment was completed, cell pellets were collected, and the procedures were performed according to the instructions of the hexokinase (HK), pyruvate kinase (PK), and glycogen assay kits. The effects of the composition of this invention on glycogen content, HK, and PK activity in IR-HepG2 cell glucose metabolism were determined, and the results are as follows: Figure 4 , 5 As shown in Figures 6 and 6 (different letters in the figure indicate significant differences), P <0.05).

[0175] from Figure 4-6 It is evident that, compared to the normal group, the glycogen content and HK and PK activities in the model group cells were significantly reduced. P <0.05%. Compared with the model group, the glycogen content and HK and PK activities of IR-HepG2 cells treated with flaxseed meal oligosaccharide extract, fraxinus ether extract, and hypoglycemic composition were significantly increased. P <0.05%, wherein the glycogen content and HK and PK activities of the hypoglycemic compositions described in Examples 7-9 were significantly higher than those of flaxseed meal oligosaccharide extract and fraxinus terpenoid extract. P The value <0.05 indicates that the hypoglycemic composition can improve the disordered glucose metabolism in IR-HepG2 cells by promoting glycogen synthesis, increasing the activity of key enzymes in glycolysis, and accelerating glucose utilization. It is superior to any component of the extract of flaxseed meal oligosaccharides or the extract of fraxinus chinensis ether terpenoids.

[0176] Experiment 3: Hypoglycemic effect of the combined compound in a diabetic mouse model

[0177] (1) Effects on fasting blood glucose in diabetic mice

[0178] Experimental Methods: Male C57BL / 6J mice (18-20g) aged 6-8 weeks were used. After 7 days of acclimatization, they were randomly divided into a control group (n=12) and a model group (n=70). The control group mice were fed a normal diet, while the model group mice were fed a high-fat diet. After 4 weeks of feeding, the model group mice were intraperitoneally injected with streptozotocin (STZ) solution (50mg / kg) for 5 consecutive days. One week later, fasting blood glucose was measured by tail vein blood collection. The criterion for successful modeling of diabetic mice was: fasting blood glucose >11.1mmol / L. The successfully modeled mice were divided into groups of 12 mice each. The specific groupings were as follows: control group: administered physiological saline by gavage; model group: administered physiological saline by gavage; low-dose group (Example 9): administered 125mg / kg by gavage; medium-dose group (Example 9): administered 250mg / kg by gavage; high-dose group (Example 9): administered 500mg / kg by gavage; positive control group (metformin): administered 250mg / kg by gavage. All experimental procedures followed the SPF animal housing guidelines, with clean bedding changed daily for the mice. The mice were administered medication via gavage once daily at 9:00 AM for four consecutive weeks. Fasting blood glucose levels were measured before administration (day 1 after successful model establishment), and at 1, 2, 3, and 4 weeks post-administration. One day before the end of the experiment, all mice were fasted for 12 hours but allowed free access to water. Blood was collected via enucleation, and the mice were euthanized, dissected, and their liver, colon, and other tissues were collected for later use.

[0179] Result: As Figure 7 As shown in the figure (different letters indicate significant differences), P <0.05), compared with the blank group, the blood glucose level of the model group mice was significantly higher than that of the blank control group ( P <0.05 indicates that a diabetic mouse model has been successfully constructed; 4 weeks after administration, the fasting blood glucose levels in the low, medium, and high dose groups of Example 9 were all lower than those in the model group ( P <0.05), with the high-dose group showing the best effect. The results indicate that the hypoglycemic composition of the present invention has a significant effect in reducing blood glucose levels in diabetic mice and alleviating diabetic symptoms.

[0180] (2) Oral glucose tolerance test

[0181] The glucose tolerance test (AGG) is a method used to assess pancreatic β-cell function and the body's ability to regulate blood glucose. As a diagnostic tool for diabetes, this test is widely used in clinical practice. The smaller the AUGG value, the faster the blood glucose level recovers.

[0182] Experimental Methods: An oral glucose tolerance test (OGTT) was conducted one day before the end of the animal experiment. Mice in each group were fasted for 12 hours, but water was allowed. Initial blood glucose levels (0 min) were measured using a glucometer. All mice were then administered glucose solution (2 g / kg) by gavage, and blood glucose levels were measured at 30, 60, and 120 min. The area under the blood glucose curve (AUC) was calculated for each group of mice.

[0183] Result: As Figure 8 As shown in the figure (different letters indicate significant differences), P <0.05), compared with the model group, the blood glucose levels of mice in the low, medium, and high dose groups and the positive control group of Example 3 were significantly reduced 120 min after oral glucose administration. In Example 9, the blood glucose AUC of the medium and high dose groups was significantly reduced ( P <0.05, and the therapeutic effect of the high-dose group was comparable to that of the positive drug group ( P >0.05). This indicates that the composition of the present invention can significantly improve the symptoms of impaired glucose tolerance in diabetic mice and stabilize blood glucose levels.

[0184] (3) Serum-related marker detection

[0185] Experimental Methods: Whole blood was allowed to stand at low temperature for 4 hours, then centrifuged at 3500 rpm for 10 minutes at 4°C. Serum was collected and stored at -80°C for later use. Fasting insulin (INS) levels were detected using an enzyme-linked immunosorbent assay (ELISA) kit, and the HOMA-IR index was calculated. Mice were tested using an automated biochemical analyzer to detect blood lipid levels and related liver and kidney function indicators, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), albumin (ALB), urea (UREA), total protein (TP), and creatinine (CRE). Serum insulin (INS) levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit. The HOMA-IR index was calculated.

[0186] Results of the effect on insulin in diabetic mice: Figure 9 As shown in the figure (different letters indicate significant differences), P <0.05), compared with the blank group, the INS level and HOMA-IR index of the model group were significantly increased ( P <0.05. This indicates that the model group mice developed insulin resistance, and their bodies were unable to actively regulate blood glucose levels. Compared with the model group, the INS levels and HOMA-IR values ​​of the low, medium, and high dose groups and the positive control group in Example 9 were all significantly reduced ( P <0.05). This indicates that the hypoglycemic composition can significantly reduce serum insulin levels in diabetic mice, improve insulin resistance, and increase insulin sensitivity in diabetic mice.

[0187] Results of the effects on serum biochemical parameters in diabetic mice: As shown in Table 2, compared with the blank group, the serum levels of TG, TC, LDL-C, ALT, AST and creatinine in the model group mice were significantly increased (P <0.05), HDL-C, ALB, and urea levels decreased significantly ( P <0.05 indicates that diabetic mice exhibit hyperlipidemia and liver and kidney dysfunction; compared with the model group, the levels of TG, TC, LDL-C, ALT, AST, and creatinine in the high-dose group and the positive control group of Example 9 were significantly reduced ( P <0.05), HDL-C, ALB, and urea levels were all significantly elevated ( P <0.05). This indicates that the hypoglycemic composition can effectively improve blood lipid levels and liver and kidney function in diabetic mice.

[0188] Table 2. Effects of the composition of the present invention on serum biochemical indicators in diabetic mice.

[0189]

[0190] Note: For peer data, different letters indicate significant differences. P <0.05, identical letters or no letters indicate no significant difference ( P >0.05).

[0191] (4) Detection of liver antioxidant indicators

[0192] Experimental methods: The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA) in the liver were determined using a kit method.

[0193] Results: As shown in Table 3, compared with the blank group, the levels of SOD, GSH-Px and CAT in the liver of mice in the model group were significantly reduced ( P <0.05), while MDA levels increased significantly ( P <0.05 indicates oxidative damage to the liver in diabetic mice. Compared with the model group, the levels of CAT, GSH-Px, and SOD were significantly increased in the medium- and high-dose groups and the positive control group in Example 9 ( P <0.05), while MDA levels decreased significantly ( P <0.05). This indicates that the composition of the present invention can enhance the antioxidant capacity of the liver in diabetic mice.

[0194] Table 3. Effects of the compositions of the present invention on the antioxidant capacity of the liver in diabetic mice.

[0195]

[0196] Note: For peer data, different letters indicate significant differences. P <0.05, identical letters or no letters indicate no significant difference ( P>0.05).

[0197] (5) Intestinal flora assay

[0198] Experimental methods: Approximately 2g of mouse cecal contents were collected into 5mL EP tubes and stored at -80℃. The 16S rDNA V3-V4 region of the samples was sequenced to analyze the diversity and richness of the gut microbiota. The detection was performed by Shanghai Paisenno Biotechnology Co., Ltd.

[0199] Results: The results are as follows Figure 10 As shown in AD. Figure 10 As shown in Figure A, the alpha diversity index dilution curve gradually flattens as the number of sequences increases, indicating that the amount of sequencing data meets the analysis requirements. Compared with the blank group, the model group and the positive control group mice had the lowest species richness; after ingesting different doses of the composition of the present invention, the abundance of intestinal microorganisms in mice was increased, indicating that the composition of the present invention can improve the abundance of intestinal microorganisms in diabetic mice. In order to comprehensively assess the alpha diversity of the microbial community, the Chao1 and Observed species indices were used to characterize the richness of intestinal microbiota, and the Shannon and Simpson indices were used to characterize the diversity of intestinal microbiota. As shown in Table 10C, compared with the model group, the Chao1, Observed species, Shannon, and Simpson indices of intestinal microbiota in the low, medium, and high dose groups of Example 9 were all significantly increased ( P <0.05). Further explanation is that the hypoglycemic composition of this invention can increase the richness and diversity of the gut microbiota in diabetic mice.

[0200] To investigate the differences in gut microbiota among different groups and to determine the impact of different treatments on gut microbiota structure, we conducted a Beta diversity analysis. Principal Coordinate Analysis (PCoA) results ( Figure 10 B) shows that the model group is farther from the control group, indicating a significant difference in gut microbiota between the two groups. Compared to the model group, the positive control group is closer to the model group, while the low and high dose groups in Example 9 are farther away, showing a trend towards the control group when viewed against the vertical axis. This demonstrates that the composition of the present invention can improve the gut microbiota structure in diabetic mice.

[0201] At the gate level ( Figure 10 On D), the most abundant phyla in the mouse cecal flora are Firmicutes (…). Firmicutes Bacteroidetes ( Bacteroidetes ) and Actinobacteria ( ActinobacteriotaFirmicutes and Bacteroides account for over 90% of the total microbial count in feces. Studies have shown that the ratio of Bacteroides to Firmicutes in feces is considered a biological indicator of human diseases such as obesity and diabetes, and is related to human health, showing a positive correlation with impaired glucose tolerance. Therefore, in diabetic and obese individuals, a higher ratio of Bacteroides to Firmicutes is better. In the control group, the ratios of Bacteroides and Firmicutes were 32.4% and 64.2%, respectively, with a ratio of 0.51. In the model group, and the low, medium, and high dose mice of Example 9 and the positive control group, the ratios of Bacteroides to Firmicutes in the gut microbiota were 0.01, 0.07, 0.19, 0.30, and 0.18, respectively. Compared with the control group, the proportion of Bacteroides in the feces of mice in the model group was significantly reduced ( P <0.05, the ratio of Bacteroidetes to Firmicutes was significantly reduced ( P <0.05). After gavage administration of the composition of the present invention, the ratio of Bacteroidetes to Firmicutes in the intestines of diabetic mice was significantly increased, with the high-dose group showing the best effect. This indicates that the hypoglycemic composition of the present invention has a positive effect on improving the gut microbiota structure of diabetic mice.

[0202] In summary, the hypoglycemic composition of this invention achieves its hypoglycemic effect by improving insulin resistance, reducing oxidative stress, and regulating gut microbiota structure. Furthermore, the hypoglycemic composition of this invention can also improve blood lipid levels and liver and kidney damage in diabetic mice, demonstrating good efficacy in treating diabetic complications.

[0203] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a flaxseed meal oligosaccharide extract with blood sugar regulating properties, characterized in that, Includes the following steps: S1. Flaxseed meal is dried, pulverized, and sterilized, and used as the extraction raw material. The extraction raw material is mixed evenly with an aqueous solution containing fermentation bacteria at a mass ratio of 1:4-7, and fermented at 25-35℃ for 24-72 hours to obtain the fermentation product; wherein the concentration of fermentation bacteria in the aqueous solution containing fermentation bacteria is 3×10⁻⁶. 6 -6×10 7 CFU / mL; S2. Add water to dilute the fermentation product obtained in step S1, and extract it by stirring at a constant temperature of 85-100℃ for 1-3 hours under normal pressure and closed environment. Then, separate the solid and liquid, take the liquid, concentrate it to a mass-volume fraction of 0.3-0.5 kg / L, and separate the supernatant. S3. Add ethanol or an aqueous ethanol solution to the product obtained in step S2, mix well, and obtain a mixed solution containing 75-85 vol% ethanol. Let the mixed solution stand at 3-6℃ for 20-30 h, separate the solid and liquid, take the solid, add water to dissolve, and obtain an aqueous extract solution. S4. Add enzyme to the aqueous extract obtained in step S3, heat to 40-60℃ for enzymatic hydrolysis for 3-6 hours, then inactivate the enzyme, separate the solid and liquid, collect the liquid, and after dialysis, concentrate and dry to obtain flaxseed meal oligosaccharide extract; wherein, the amount of enzyme added is 2-5% of the mass of the raw material extracted in step S1; The fermenting bacteria mentioned in step S1 is *Saccharomyces cerevisiae*. The preparation steps of the aqueous solution containing the fermenting bacteria are as follows: Take *Saccharomyces cerevisiae* strain as the strain to be activated, dilute the strain to be activated with sterile water or glucose liquid medium, inoculate it onto glucose solid medium, and incubate at 25-35℃ for 48-72 h. Then, use an inoculation loop to pick mature, single colonies and inoculate them into glucose liquid medium. After shaking culture at 25-35℃ for 24-30 h, adjust the bacterial concentration to 1×10⁻⁶. 8 -1×10 9 CFU / mL was used to obtain a fermentation suspension. The fermentation suspension was then diluted with water to obtain an aqueous solution containing the fermentation bacteria. The enzymes mentioned in step S4 were selected from cellulase, pectinase, or xylanase.

2. The method for preparing the flaxseed meal oligosaccharide extract with blood sugar regulating properties according to claim 1, characterized in that: When diluting with water in step S2, the amount of water used is 8-12 times the weight of the extracted raw material; the volume fraction of ethanol in the ethanol-water solution in step S3 is 90-95%; when dissolving with water in step S3, the amount of water used is 1-1.5 times the mass of the extracted raw material in step S1.

3. The method for preparing the flaxseed meal oligosaccharide extract with blood sugar regulating properties according to claim 1, characterized in that: The sterilization in step S1 is carried out at 0.08-0.12 MPa and 120-125℃ for 15-25 min; the separation of supernatant in step S2 and the solid-liquid separation in step S3 are both carried out by centrifugation, with the centrifugation speed controlled at 4500-5500 rpm and the centrifugation time at 8-12 min; the solid-liquid separation in steps S2 and S4 is carried out by filtration, and the concentration in steps S2 and S4 is carried out by vacuum concentration; the enzyme inactivation in step S4 is carried out by boiling water bath for 4-6 min; the drying in step S4 is carried out by freeze drying, and the freeze drying conditions are: vacuum degree 10-50 Pa, temperature -20--40℃, and time 24-48 h.

4. A flaxseed meal oligosaccharide extract with blood sugar regulating properties prepared by any one of claims 1 to 3, characterized in that: The oligosaccharide content of flaxseed meal oligosaccharide extract is over 92%, and the molecular weight of flaxseed meal oligosaccharide extract is 300-2000 Da.

5. A hypoglycemic composition, characterized in that, The raw materials include the following parts by weight: 4-9 parts of the flaxseed meal oligosaccharide extract as described in claim 4, and 3-6 parts of the fraxinus chinensis ether terpene extract.

6. The hypoglycemic composition according to claim 5, characterized in that, The extract of fraxinus chinensis cyclohexene ethers and terpenoids is prepared by the following steps: The pulverized and sieved fraxinus chinensis is dispersed in DES aqueous solution at a material-to-liquid ratio of 1:(20-40) g / mL to obtain a mixed solution; cellulase is added to the mixed solution, and after mixing, it is extracted by microwave at 40-60℃ for 30-60 min, filtered, and the filtrate is concentrated to a mass-volume fraction of 0.3-0.5 kg / L. The supernatant is separated and injected into an adsorption resin. First, water is used to elute until the eluent is nearly colorless, and the eluent is discarded. Then, 20-40 vol% ethanol aqueous solution is used to elute and remove impurities, and the eluent is discarded. Finally, 80-90 vol% ethanol aqueous solution is used to elute, and the eluent of 80-90 vol% ethanol aqueous solution is collected, concentrated under reduced pressure, and freeze-dried to obtain the final product. The DES aqueous solution is prepared by mixing DES solvent and water, with the water content in the DES aqueous solution being 10-30% by mass. The DES solvent is prepared by mixing choline chloride and carboxylic acid or choline chloride and polyol in a molar ratio of 1:2-3. The amount of cellulase added is 2-5% of the mass of Fraxinus chinensis bark.

7. The hypoglycemic composition according to claim 6, characterized in that: The carboxylic acid is selected from oxalic acid, and the polyol is selected from 1,4-butanediol or glycerol; during microwave extraction, the microwave power is 450-550W, and the supernatant is separated by centrifugation, with the centrifugation speed controlled at 4500-5500 rpm and the centrifugation time at 8-12 min; the freeze-drying conditions are: vacuum degree 10-50 Pa, temperature -20 to 40℃, and time 24-48 h.

8. The hypoglycemic composition according to claim 6, characterized in that: The adsorption resin used is a macroporous resin of type AB-8, D101, or NKA-9, and the amount of adsorption resin used is 1-1.5 times the mass of the Fraxinus bark. When eluting with water, the amount of water used is 5-8 times the mass of the Fraxinus bark, and the elution rate is 1.0-2.0 Bv / h. When eluting with 20-40 vol% ethanol aqueous solution, the amount of 20-40 vol% ethanol aqueous solution used is 5-8 times the mass of the Fraxinus bark, and the elution rate is 1.0-2.0 Bv / h. When eluting with 80-90 vol% ethanol aqueous solution, the amount of 80-90 vol% ethanol aqueous solution used is 9-12 times the mass of the Fraxinus bark, and the elution rate is 1.0-2.0 Bv / h.

9. The use of the hypoglycemic composition according to any one of claims 5 to 8 in the preparation of a hypoglycemic medicament.

Images