A method for separating and purifying barley flavonoid monomers and saponarin monomers and application thereof

Abstract

The present application belongs to the technical field of separation and purification, and particularly relates to a separation and purification method and application of barley flavonoid monomer and saponarin monomer. The raw material containing the barley flavonoid and the saponarin is subjected to heating reflux extraction, AB-8 macroporous resin enrichment and medium-pressure preparation chromatography separation technology, so that the barley flavonoid monomer and the saponarin monomer can be obtained. No solvent system harmful to the environment and the human body is used, the operation is simple, the industrialized production is easy to realize, the repeatability and stability are good, the purity of the obtained barley flavonoid monomer is 98.5% to 99.9%, and the purity of the saponarin monomer is 98.4% to 99.9%. The biological experiment shows that the obtained barley flavonoid monomer and saponarin monomer have alpha-glucosidase inhibitory activity, can slow down the absorption of glucose in the intestinal tract, have obvious blood sugar lowering effect, and can be used for preparing blood sugar lowering, preventing and / or treating diabetes products.

Description

Technical Field

[0001] This invention belongs to the field of separation and purification technology, specifically relating to a method and application for the separation and purification of barley flavin monomer and saponin monomer. Background Technology

[0002] Flavonoids are a class of highly pharmacologically active natural compounds with various biological activities, including anti-cardiovascular and cerebrovascular disease, anti-tumor, antioxidant, and anti-inflammatory effects. Barley seedlings are the young shoots of barley (Hordeum vulgare L.), a plant belonging to the genus Hordeum in the family Poaceae. They are characterized by "three highs and two lows": high protein (high amino acids), high dietary fiber, high vitamins, low fat, and low sugar. The two main types of flavonoids in barley seedlings are horvinoside and saponin.

[0003] Separating and purifying organic mixtures is a crucial preliminary step in organic structure research. Typically, obtaining a certain amount of high-purity organic monomers is a prerequisite for structural and pharmacological analysis of organic compounds. When the organic monomers in a mixture are highly similar in structure and properties, their separation and purification become extremely difficult. Common problems include obtaining only a single-component product, low purity of the obtained product, the use of solvent systems harmful to the environment and human health, complex solvent systems, high production costs, and small-scale production that cannot meet market demand. For example, Chinese patent CN101696381A discloses a new process for preparing barley flavonoid extract and its application in health wine. This process involves hot reflux extraction of barley seedlings with an alcohol solution, followed by clarification using a ceramic membrane, separation using an ultrafiltration membrane, and concentration using a nanofiltration membrane to obtain the barley flavonoid extract. This extract contains two components, phytoflavin and saponin, which cannot be separated. Summary of the Invention

[0004] The purpose of this invention is to provide a method and application for separating and purifying phytoflavin monomers and saponin monomers, obtaining high-purity phytoflavin and saponin monomers with good repeatability and stability, and exploring the medical applications of phytoflavin and saponin monomers.

[0005] This invention provides a method for separating and purifying phytoflavin and saponin monomers, comprising the following steps:

[0006] (1) Mix the raw materials containing phytoflavin and saponin with solvent, heat and reflux to extract, concentrate the extract to obtain crude extract;

[0007] The solvent is water or an aqueous ethanol solution; the volume concentration of ethanol in the aqueous ethanol solution is >0 and ≤40%; when the solvent is an aqueous ethanol solution, the ethanol in the extract is separated and then concentrated to obtain a crude extract;

[0008] (2) After dissolving the crude extract in hot water at 70°C, it was adsorbed onto AB-8 macroporous resin. After complete adsorption, it was eluted with water until colorless, and then eluted with an ethanol aqueous solution with a volume concentration of 10-50%. The ethanol eluent was collected, concentrated, and dried to obtain the total flavonoid extract.

[0009] (3) After dissolving the total flavonoid extract in an ethanol aqueous solution with a volume concentration of 10-40%, the extract is separated by medium-pressure preparative chromatography. Under the detection condition of 340 nm wavelength, the eluents of barley flavonoid monomer and saponin flavonoid monomer are collected according to the chromatogram. The step of separation by medium-pressure preparative chromatography includes isocratic elution using an ethanol aqueous solution with a volume concentration of 10-30% as the mobile phase.

[0010] (4) Separate the ethanol from the eluent of the barley flavonoid monomer and the eluent of the saponin monomer, and dry them to obtain barley flavonoid monomer and saponin monomer respectively.

[0011] Preferably, the heating and reflux extraction is performed 1 to 3 times, and the heating and reflux extraction time is 1 to 3 hours each time; the heating and reflux extraction temperature is above 90°C.

[0012] Preferably, during each heating and reflux extraction, the ratio of the raw material containing phytoflavin and saponin to the ethanol aqueous solution is 1g:20-30mL.

[0013] Preferably, the raw materials containing phytoflavin and saponin include barley seedlings and / or barley seedlings.

[0014] Preferably, the loading rate of the AB-8 macroporous resin is 50-200 L / h;

[0015] The adsorption time is ≥24h.

[0016] Preferably, the elution with water is performed by eluting with 1-5 times the column volume of water; the water flow rate is 100-200 L / h.

[0017] Preferably, the elution with an ethanol aqueous solution with a volume concentration of 10-50% is performed by eluting with 3-6 column volumes of an ethanol aqueous solution with a volume concentration of 10-50%; the flow rate of the ethanol aqueous solution is 100-200 L / h.

[0018] The present invention also provides the application of barley flavin monomer and / or saponin flavin monomer obtained by the separation and purification method described above in the preparation of hypoglycemic products;

[0019] The purity of the phytoestrogen monomer in the product is 98.5% to 99.9%;

[0020] The purity of saponin monomer in the product is 98.4% to 99.9%.

[0021] The present invention also provides the application of barley flavin monomer and / or saponin flavin monomer obtained by the separation and purification method described above in the preparation of products for the prevention and / or treatment of diabetes;

[0022] The purity of the phytoestrogen monomer in the product is 98.5% to 99.9%;

[0023] The purity of saponin monomer in the product is 98.4% to 99.9%.

[0024] Beneficial effects:

[0025] This invention involves heating and reflux extraction of raw materials containing physcion and saponin, followed by AB-8 macroporous resin lipid enrichment and medium-pressure preparative chromatography to separate and purify the extract, yielding physcion monomers and saponin monomers. The separation and purification method provided by this invention does not use solvent systems harmful to the environment and human body, is simple to operate, easy to implement in industrial production, and exhibits good repeatability and stability. The purity of the obtained physcion monomer is 98.5%–99.9%, and the purity of the saponin monomer is 98.4%–99.9%. Biological experiments show that the obtained physcion and saponin monomers possess α-glucosidase inhibitory activity, which can slow down the absorption of glucose in the intestine and has a significant hypoglycemic effect. They can be used to prepare products for lowering blood sugar and preventing and / or treating diabetes. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0027] Figure 1 This is a liquid chromatogram of the crude extract;

[0028] Figure 2 The image shows the liquid chromatogram of the total flavonoid extract.

[0029] Figure 3 To collect liquid chromatograms of the separation of barley flavin monomer and saponin monomer;

[0030] Figure 4 This is a liquid chromatogram of phytoflavin monomers;

[0031] Figure 5 This is a liquid chromatogram of saponin monomer;

[0032] Figure 6 The chemical structural formulas of barley flavin monomer and saponin flavin monomer are shown.

[0033] Figure 7 This is a standard curve for α-glucosidase activity.

[0034] Figure 8 The results show the inhibition type of α-glucosidase by barley flavin monomer;

[0035] Figure 9 The results show the inhibition type of α-glucosidase by saponin monomer. Detailed Implementation

[0036] This invention provides a method for separating and purifying phytoflavin and saponin monomers, comprising the following steps:

[0037] (1) Mix the raw materials containing phytoflavin and saponin with solvent, heat and reflux to extract, concentrate the extract to obtain crude extract;

[0038] The solvent is water or an aqueous ethanol solution; the volume concentration of ethanol in the aqueous ethanol solution is >0 and ≤40%; when the solvent is an aqueous ethanol solution, the ethanol in the extract is separated and then concentrated to obtain a crude extract;

[0039] (2) After dissolving the crude extract in hot water at 70°C, it was adsorbed onto AB-8 macroporous resin. After complete adsorption, it was eluted with water until colorless, and then eluted with an ethanol aqueous solution with a volume concentration of 10-50%. The ethanol eluent was collected, concentrated, and dried to obtain the total flavonoid extract.

[0040] (3) After dissolving the total flavonoid extract in an ethanol aqueous solution with a volume concentration of 10-40%, the extract is separated by medium-pressure preparative chromatography. Under the detection condition of 340 nm wavelength, the eluents of barley flavonoid monomer and saponin flavonoid monomer are collected according to the chromatogram. The step of separation by medium-pressure preparative chromatography includes isocratic elution using an ethanol aqueous solution with a volume concentration of 10-30% as the mobile phase.

[0041] (4) Separate the ethanol from the eluent of the barley flavonoid monomer and the eluent of the saponin monomer, and dry them to obtain barley flavonoid monomer and saponin monomer respectively.

[0042] This invention involves mixing raw materials containing phytoflavin and saponin with a solvent, heating and refluxing for extraction, and concentrating the resulting extract to obtain a crude extract.

[0043] In this invention, the solvent is preferably water or an aqueous ethanol solution; the volume concentration of ethanol in the aqueous ethanol solution is preferably >0 and ≤40%, more preferably 10% to 30%. When the solvent is an aqueous ethanol solution, this invention preferably separates the ethanol from the extract and then concentrates it to obtain a crude extract.

[0044] In this invention, the number of reflux extractions is preferably 1 to 3 times, more preferably 2 times; the temperature of the reflux extraction is preferably above 90°C, more preferably 90°C. The time for each reflux extraction is preferably 1 to 3 hours, more preferably 2 hours. The extracts from each reflux extraction are combined. The concentration method is preferably vacuum concentration.

[0045] In this invention, the ratio of the raw material containing phytoflavin and saponin to the ethanol aqueous solution is 1g:20-30mL, more preferably 1g:25mL. The raw material containing phytoflavin and saponin preferably includes barley seedlings and / or barley seedlings.

[0046] After obtaining the crude extract, the present invention dissolves the crude extract in hot water at 70°C and then adsorbs it onto AB-8 macroporous resin. In this invention, the adsorption time is preferably ≥24 hours, more preferably 24 hours. The loading rate onto the AB-8 macroporous resin is preferably 50-200 L / h, more preferably 100 L / h. The present invention preferably removes impurities from the dissolved crude extract, obtaining a clear liquid, and then adsorbs it onto AB-8 macroporous resin; the impurity removal method is preferably tubular centrifugation using a GQ75 high-speed tubular separator.

[0047] After complete adsorption, the present invention elutes with water until colorless, preferably with 1-5 column volumes of water, and more preferably with 3 column volumes of water. In the present invention, the flow rate of the water is preferably 100-200 L / h, more preferably 200 L / h; the water is preferably deionized water.

[0048] After elution with water, the present invention elutes with an ethanol-water solution of 10-50% (v / v), preferably 30% (v / v), collects the ethanol eluent, concentrates it, and dries it to obtain the total flavonoid extract. The present invention preferably elutes with 3-6 column volumes of ethanol-water solution, more preferably with 5 column volumes of ethanol-water solution; the flow rate of the ethanol-water solution is preferably 100-200 L / h, more preferably 150 L / h. The concentration method of the present invention preferably includes sequential vacuum concentration. The drying method of the present invention is preferably freeze drying.

[0049] After obtaining the total flavonoid extract, the present invention uses a 10-40% (v / v) ethanol aqueous solution, preferably a 20% (v / v) ethanol aqueous solution, to dissolve the total flavonoid extract. Separation is performed using medium-pressure preparative chromatography. Based on the chromatogram under 340 nm wavelength detection conditions, the eluents of phytoflavin monomer and saponin monomer are collected separately. In the present invention, the step of separation using medium-pressure preparative chromatography preferably includes isocratic elution using a 10-30% (v / v) ethanol aqueous solution, preferably a 22% (v / v) ethanol aqueous solution, as the mobile phase.

[0050] After obtaining the eluents of barley flavin monomer and saponin monomer, the present invention separates the ethanol from the eluents of barley flavin monomer and saponin monomer, and dries them to obtain barley flavin monomer and saponin monomer. The preferred method for separating the ethanol in the present invention is vacuum concentration; the preferred method for drying is freeze drying.

[0051] The separation and purification method provided by this invention does not use solvent systems that are harmful to the environment and human body. It is simple to operate, easy to implement for industrial production, and has good repeatability and stability. The purity of the obtained barley flavin monomer is 98.5% to 99.9%, and the purity of saponin monomer is 98.4% to 99.9%. Biological experiments have shown that the obtained barley flavin monomer and saponin monomer have α-glucosidase inhibitory activity, which can slow down the absorption of glucose in the intestine and has a significant hypoglycemic effect. They can be used to prepare hypoglycemic, preventive and / or therapeutic products for diabetes.

[0052] Given the effectiveness of the separation and purification method provided by this invention, the hay fever glycoside monomer with a purity of 98.5%–99.9% and the saponin monomer with a purity of 98.4%–99.9% obtained after separation and purification by the above technical solution, as well as the application of the obtained hay fever glycoside monomer and / or saponin monomer in the preparation of hypoglycemic products and products for the prevention and / or treatment of diabetes, also fall within the scope of protection of this invention. In this invention, the products preferably include pharmaceuticals, health products, or functional foods. This invention does not impose strict requirements on the dosage form of the pharmaceuticals, health products, or functional foods; conventional choices are acceptable, such as solid beverages, compressed candies, capsules, microcapsules, and drop pills.

[0053] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a method for separating and purifying barley flavin monomers and saponin monomers, as well as their applications. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0054] The experimental instruments, reagents, and materials used in this invention are as follows:

[0055] Preparation and testing instruments: XS204 electronic balance (Mettler-Toledo AG, Switzerland); TCS-150 electronic platform scale (Zhejiang Yuyang Weighing Instrument Co., Ltd.); QJY-200 straight-cut medicine cutter (Shanghai Nanjue Traditional Chinese Medicine Machinery Manufacturing Co., Ltd.); R-1020 rotary evaporator (Zhengzhou Changcheng Science & Industry Trade Co., Ltd.); TQG-0.3-01 extraction tank (Zhejiang Wenxiong Machinery Valve Co., Ltd.); SJN-150-00 double-effect concentrator (Zhejiang Wenxiong Machinery Valve Co., Ltd.); GQ75 high-speed tubular separator. Centrifuge (Shanghai Pudong Tianben Centrifuge Machinery Co., Ltd.); SZ-50-01 macroporous resin column (Zhejiang Wenxiong Machinery Valve Industry Co., Ltd.); DHG-9140A electric thermostatic drying oven (Shanghai Yiheng Technology Co., Ltd.); DAC-HB100 medium-pressure preparative liquid chromatograph (Jiangsu Hanbang Technology Co., Ltd.); LC3100 high-performance liquid chromatograph (Anhui Wanyi Co., Ltd.); SYH type laboratory three-dimensional motion mixer (Jiangsu Chitong Machinery Manufacturing Co., Ltd.); Encapsulator B-390 microcapsule granulator (BUCHI); GL2-25 laboratory dry granulator (Jiangsu Zhangjiagang Kaichuang Machinery Manufacturing Co., Ltd.); NJP-2-200C fully automatic capsule filling machine (Ruian Tianhong Pharmaceutical Machinery Co., Ltd.); ZPSX10 rotary tablet press (Shanghai Xiangshun Pharmaceutical Machinery Co., Ltd.); GLZY-0.5B freeze dryer (Shanghai Pudong Freeze Drying Equipment Co., Ltd.); Agilent 1260 high-performance liquid chromatograph (Agilent Technologies, Inc., USA);

[0056] Instruments used for drug efficacy testing: METTLERTOLEDO MS205DU and PL602-L electronic balances (Mettler-Toledo, Switzerland); blood glucose meter and blood glucose test strips (Beijing Yicheng Bioelectronics Technology Co., Ltd.); high-speed refrigerated centrifuge (Shanghai Jiangdong Instrument Factory); fully automated microplate reader (Shanghai Lenovo Biotechnology Co., Ltd.); BPH-9162 precision constant temperature cell culture incubator (Shanghai Huitai Instrument Manufacturing Co., Ltd.); SW-CJ-1FD ultra-clean workbench (Sujing Antai Air Technology Co., Ltd.); inverted microscope (Leica Instruments, Germany); pipettes (Eppendorf, Germany); disposable 96-well plates (Corning); pH 35 pen-type pH meter (Shanghai Shunyu Hengping Scientific Instruments Co., Ltd.); EYELA constant temperature water bath (Shanghai Ailang Instruments Co., Ltd.);

[0057] Reagents and Materials: Barley seedlings, collected from Liushuzhuang Village, Chengguan Town, Datong County, Qinghai Province; AB-8 macroporous resin (Hebei Cangzhou Baoen Adsorption Materials Technology Co., Ltd.); methanol for HPLC analysis was chromatographically pure (Shandong Yuwang Company); ultrapure water (18.25 MΩ) was prepared using the ULUPURE ultrapure water system; all other reagents were analytical grade and purchased from Tianjin Fuyu Chemical Co., Ltd. (Tianjin, China); streptozotocin (Sigma); 4-nitrophenyl-α-D-glucopyranoside (PNPG, Sigma); acarbose (Beijing Lanbolide Trading Co., Ltd.); metformin hydrochloride (Jinan Hengji Pharmaceutical Co., Ltd.); citric acid (Tianjin Baishi Chemical Co., Ltd.); sodium citrate (Tianjin Yongda Chemical Reagent Co., Ltd.); disodium hydrogen phosphate (Shandong Laiyang Shuangshuang Chemical Co., Ltd.); sodium dihydrogen phosphate (Tianjin Damao Chemical Reagent Manufacturing Co., Ltd.); dimethyl sulfoxide (Tianjin... Kaitong Chemical Reagent Co., Ltd.; Glucose Assay Kit (Shanghai Rongsheng Biopharmaceutical Co., Ltd.); Insulin (INS) Kit, Cholesterol (TC) Kit, Triglyceride (TG) Kit, High-Density Lipoprotein (HDL) Kit, Low-Density Lipoprotein (LDL) Kit, Superoxide Dismutase (SOD) Kit, Malondialdehyde (MDA) Kit, all purchased from Wuhan Genemei Biotechnology Co., Ltd.; High-fat, high-sugar feed (Nanjing Shengmin Scientific Animal Farm); Fetal bovine serum (BI); Dexamethasone, insulin, berberine (Shanghai Maclean Biochemical Technology Co., Ltd.); DMEM high- and low-sugar culture media, PBS, trypsin, double antibiotics (Gibco); 3-Isobutyl-1-methylxanthine (IBMX, Sigma); Red Oil O staining solution (Nanjing Jiancheng Biotechnology Co., Ltd.); Glucose Assay Kit (Shanghai Rongsheng Biopharmaceutical Co., Ltd.);

[0058] Cells: 3T3-L1 preadipocytes, Kunming Cell Bank, Chinese Academy of Sciences; Animals: SPF-grade male SD rats, provided by the Experimental Animal Center of Xi'an Jiaotong University School of Medicine, batch number: 61001700002271, 200±20g, 100 rats in total.

[0059] Example 1

[0060] The method for separating and purifying phytoflavin and saponin monomers consists of the following steps:

[0061] (1) Extraction: 10 kg of dried barley seedlings were chopped using a QJY-200 straight-cutting herb cutter. 300 L of 30% ethanol aqueous solution was added at a ratio of 1 g: 30 mL. The mixture was then subjected to hot reflux extraction in a TQG-0.3-01 extraction tank at a temperature of 90 °C for 3 extractions, each lasting 2 h. The extracts were combined and concentrated under reduced pressure using an SJN-150-00 double-effect concentrator to recover the ethanol, yielding 12 kg of crude extract (80% water content).

[0062] The crude extract was analyzed using an Agilent 1260 high-performance liquid chromatograph (HPLC), and the results are as follows: Figure 1 As shown, the chromatographic column was a Platisil ODS (250×4.6mm, 5μm) column, the column temperature was 30℃, the mobile phase was methanol-water, the gradient elution was 0-30min, 10-45% methanol, the detection wavelength was 340nm, and the flow rate was 1.0mL / min.

[0063] according to Figure 1 It can be seen that the barley seedling extract mainly contains two compounds, compound 1 (barley flavin) and compound 2 (saponin).

[0064] (2) Macroporous resin enrichment: After the crude extract obtained in step (1) was dissolved in water, it was centrifuged in a GQ75 high-speed tubular separator to remove impurities. The clarified liquid was adsorbed onto an AB-8 macroporous resin column at a flow rate of 100 L / h and allowed to stand for more than 24 hours. Then, it was washed with water at a flow rate of 200 L / h for 3 times the column volume, and then eluted with an ethanol aqueous solution with a volume concentration of 30% at a flow rate of 100 L / h for 5 times the column volume. The ethanol aqueous solution eluent with a volume concentration of 30% was collected and concentrated under reduced pressure using an SJN-150-00 double-effect concentrator to recover ethanol. Then, it was further concentrated using an R-1020 rotary evaporator. The concentrate was dried in a DHG-9140A electric thermostatic drying oven to obtain 480g of total flavonoid extract.

[0065] The total flavonoid extract obtained was analyzed by HPLC using an Agilent 1260 high-performance liquid chromatograph, and the results are as follows: Figure 2 As shown, the chromatographic column was a Platisil ODS (250×4.6mm, 5μm) column, the column temperature was 30℃, the mobile phase was methanol-water, the gradient elution was 0-30min, 10-45% methanol, the detection wavelength was 340nm, and the flow rate was 1.0mL / min.

[0066] according to Figure 2 It can be seen that after treatment with AB-8 macroporous resin, compounds 1 (horseflavin) and 2 (saponin) were well enriched in the total flavonoid extract.

[0067] (3) Medium-pressure preparative liquid chromatography separation: The total flavonoid extract described in step (2) was fully dissolved in 20% ethanol and then separated and purified using a DAC-HB100 medium-pressure preparative liquid chromatograph. Isocratic elution was performed using 15% ethanol as the mobile phase, and the chromatogram was analyzed under detection conditions at a wavelength of 340 nm. Figure 3 Collect the eluted fractions of phytoflavin and saponin, recover the ethanol solution, freeze-dry, and obtain 38g of phytoflavin monomer and 12g of saponin monomer.

[0068] The obtained phytoflavin monomers and saponin monomers were analyzed by HPLC using an Agilent 1260 high-performance liquid chromatograph. The results are as follows: Figures 4-5 As shown, the chromatographic column was a Platisil ODS (250×4.6mm, 5μm) column, the column temperature was 30℃, the mobile phase was methanol-water, the gradient elution was 0-30min, 10-45% methanol, the detection wavelength was 340nm, and the flow rate was 1.0mL / min.

[0069] according to Figures 4-5 It can be seen that after medium-pressure preparative liquid phase treatment, phytoflavin and saponin monomers were separated, with phytoflavin monomer content of 99.9% by weight and saponin monomer content of 98.6% by weight.

[0070] The structural formula of lutonarin monomer is as follows: Figure 6 1. NMR data are shown in Table 1; the structural formula of saponarin monomer is shown in Table 1. Figure 6 Table 2 shows the NMR data.

[0071] Table 1. NMR data of phytoflavin

[0072]

[0073]

[0074] Table 2. NMR data of saponins

[0075]

[0076]

[0077] Example 2

[0078] The method for separating and purifying phytoflavin and saponin monomers consists of the following steps:

[0079] (1) Extraction: 10 kg of dried barley seedlings were chopped using a QJY-200 straight-cutting herb cutter. 200 L of 10% ethanol was added at a ratio of 1 g: 20 mL. The mixture was then subjected to hot reflux extraction in a TQG-0.3-01 extraction tank at 90 °C for 2 hours each time. The extracts were combined and concentrated under reduced pressure using an SJN-150-00 double-effect concentrator to recover the ethanol, yielding 10.5 kg of crude extract (82% water content).

[0080] (2) Macroporous resin enrichment: The crude extract obtained in step (1) was dissolved in water and centrifuged in a GQ75 high-speed tubular separator to remove impurities. The clarified liquid was adsorbed onto an AB-8 macroporous resin column at a flow rate of 50 L / h and allowed to stand for more than 24 hours. Then, it was washed with 5 column volumes of water at a flow rate of 100 L / h and eluted with 4 column volumes of 20% ethanol at a flow rate of 100 L / h. The 20% ethanol eluent was collected and concentrated under reduced pressure using an SJN-150-00 double-effect concentrator to recover the ethanol. The concentrate was then further concentrated using an R-1020 rotary evaporator. The concentrate was dried in a DHG-9140A electric thermostatic drying oven to obtain 370 g of total flavonoid extract.

[0081] (3) Medium-pressure preparative liquid chromatography separation: The total flavonoid extract described in step (2) was fully dissolved in 30% ethanol and then separated and purified using a DAC-HB100 medium-pressure preparative liquid chromatograph. Isocratic elution was performed using 25% ethanol as the mobile phase, with repeated injections. The eluted fractions of phytoflavin and saponin were collected according to the chromatogram under a detection wavelength of 340 nm. The ethanol solution was recovered, and the fractions were freeze-dried to obtain 30 g of phytoflavin and 9.5 g of saponin. HPLC analysis using an Agilent 1260 high-performance liquid chromatograph showed that the phytoflavin content was 98.8% by weight and the saponin content was 98.4% by weight.

[0082] Example 3

[0083] The method for separating and purifying phytoflavin and saponin monomers consists of the following steps:

[0084] (1) Extraction: 10 kg of dried barley seedlings were chopped using a QJY-200 straight-cutting herb cutter and 250 L of pure water was added at a ratio of 1 g: 25 mL. The mixture was then subjected to hot reflux extraction in a TQG-0.3-01 extraction tank at a temperature of 90 °C for 3 extractions, each lasting 2 h. The extracts were combined and concentrated under reduced pressure using an SJN-150-00 double-effect concentrator to recover ethanol, yielding 14 kg of crude extract (85% water content).

[0085] (2) Macroporous resin enrichment: The crude extract obtained in step (1) was dissolved in water and centrifuged in a GQ75 high-speed tubular separator to remove impurities. The clarified liquid was adsorbed onto an SZ-50-01 macroporous resin column at a flow rate of 200 L / h and allowed to stand for more than 24 hours. Then, it was washed with 3 column volumes of water at a flow rate of 200 L / h and eluted with 40% ethanol at a flow rate of 200 L / h for 4 column volumes. The 40% ethanol eluent was collected and concentrated under reduced pressure using an SJN-150-00 double-effect concentrator to recover the ethanol. The concentrate was then further concentrated using an R-1020 rotary evaporator. The concentrate was dried in a DHG-9140A electric thermostatic drying oven to obtain 420 g of total flavonoid extract.

[0086] (3) Medium-pressure preparative liquid chromatography separation: The total flavonoid extract described in step (2) was fully dissolved in 12% ethanol and then separated and purified using a DAC-HB100 medium-pressure preparative liquid chromatograph. Isocratic elution was performed using 22% ethanol as the mobile phase, with repeated injections. The eluted fractions of phytoflavin and saponin were collected according to the chromatogram under a detection wavelength of 340 nm. The ethanol solution was recovered and dried in a DHG-9140A electric thermostatic drying oven at approximately 80°C to obtain 33 g of phytoflavin and 10.5 g of saponin. HPLC analysis using an Agilent 1260 high-performance liquid chromatograph showed that the phytoflavin content was 98.5% by weight and the saponin content was 99.9% by weight.

[0087] Example 4

[0088] The method for separating and purifying phytoflavin and saponin monomers consists of the following steps:

[0089] (1) Extraction: 10 kg of dried barley seedlings were chopped using a QJY-200 straight-cutting herb cutter. 300 L of 40% ethanol was added at a ratio of 1 g: 30 mL. The mixture was then subjected to hot reflux extraction in a TQG-0.3-01 extraction tank at a temperature of 90 °C. The extraction was performed twice, for 2 h each time. The extracts were combined and concentrated under reduced pressure using an SJN-150-00 double-effect concentrator to recover the ethanol, yielding 11.5 kg of crude extract (81% water content).

[0090] (2) Macroporous resin enrichment: The crude extract obtained in step (1) was dissolved in water and centrifuged in a GQ75 high-speed tubular separator to remove impurities. The clarified liquid was adsorbed onto an AB-8 macroporous resin column at a flow rate of 150 L / h and allowed to stand for more than 24 hours. Then, it was washed with water at a flow rate of 150 L / h for 2 times the column volume, and then eluted with 25% ethanol at a flow rate of 150 L / h for 5-6 times the column volume. The 25% ethanol eluent was collected and concentrated under reduced pressure using an SJN-150-00 double-effect concentrator to recover the ethanol. The concentrate was then further concentrated using an R-1020 rotary evaporator. The concentrate was dried in a DHG-9140A electric thermostatic drying oven to obtain 450 g of total flavonoid extract.

[0091] (3) Medium-pressure preparative liquid chromatography separation: The total flavonoid extract described in step (2) was dissolved thoroughly in 30% ethanol by heating, and then separated and purified using a DAC-HB100 medium-pressure preparative liquid chromatograph. Isocratic elution was performed using 20% ​​ethanol as the mobile phase, with repeated injections. The eluted fractions of phytoflavin and saponin were collected according to the chromatogram under a detection wavelength of 340 nm. The ethanol solution was recovered, and the fractions were freeze-dried to obtain 36 g of phytoflavin and 11 g of saponin. HPLC analysis using an Agilent 1260 high-performance liquid chromatograph showed that the phytoflavin content was 99.0% by weight and the saponin content was 98.5% by weight.

[0092] Test Example 1

[0093] The therapeutic effects of barley flavin monomer and saponin flavin monomer on streptozotocin (STZ)-induced diabetic rats

[0094] 1. Establishment of a streptozotocin (STZ)-induced diabetic rat model

[0095] SD rats were acclimatized in an animal laboratory for one week, during which they had free access to regular feed and tap water. After the acclimatization period, they were randomly divided into a blank control group (n=20) and a type 2 diabetes group (n=80) based on their body weight. The blank control group was fed a regular feed, while the type 2 diabetes group was fed a high-fat, high-sugar feed. After 8 weeks, the type 2 diabetes group was fasted for 12 hours but allowed free access to water. They were then given a single intraperitoneal injection of 1% streptozotocin solution at a dose of 35 mg / kg BW. The blank control group was injected with the same volume of citrate buffer, but had free access to food and water. 72 hours after the streptozotocin injection, the rats were fasted for 12 hours but allowed free access to water. Blood was collected from the tail vein, and fasting blood glucose was measured using a glucometer for 3 consecutive days. Rats with a blood glucose level >16.7 mmol / L were considered diabetic models.

[0096] 2. Experimental Methods

[0097] 2.1 The day after successful modeling, the type 2 diabetes group was randomly divided into a barley glycoside monomer group, a saponin glycoside monomer group, a metformin hydrochloride group, and a diabetes model group. The barley glycoside monomer group was administered the barley glycoside monomer obtained in Example 1 by gavage once a day at a dose of 0.1 g / kg BW. The saponin glycoside monomer group was administered the saponin glycoside monomer obtained in Example 3 by gavage once a day at a dose of 0.1 g / kg BW. The metformin hydrochloride group was administered metformin hydrochloride by gavage once a day at a dose of 0.075 g / kg BW. The diabetes model group and the blank control group were administered an equal volume of distilled water by gavage once a day at a time. Each treatment group was administered the drug by gavage for 28 consecutive days. During the drug administration period, except for the blank control group which was given a normal diet, the other groups were given a high-fat and high-sugar diet, and all rats had free access to food and water.

[0098] 2.2. From the start of drug administration, the weight of each experimental animal was measured daily at regular intervals, with each experimental group as a unit. Sufficient feed and water were provided daily at regular intervals, and the remaining amounts were monitored at the same time the following day. The average food intake and water intake per rat per 24 hours were calculated. Urine was collected daily at regular intervals, and the average urine output per rat per 24 hours was measured and calculated. Blood was collected via the tail vein on days 0, 7, 14, 21, and 28 of drug administration, after a 12-hour fast with free access to water. Fasting blood glucose levels were measured using a glucose assay kit. During this period, the activity level, mental state, and coat color of the rats in each group were observed, and the mortality rate of rats in each experimental group was recorded.

[0099] 2.3. An oral glucose tolerance test (OGTT) was performed after the last administration. Before the experiment, rats were fasted but allowed free water for 16 hours. After fasting, fasting blood glucose levels were measured first. 5 g / kg BW 50% glucose was injected subcutaneously, and blood samples were collected at 0, 0.5 h, 1 h, and 2 h to measure blood glucose levels in the rats. The postprandial blood glucose levels of STZ-induced diabetic rats in different treatment groups were analyzed.

[0100] 2.4. After the last administration, the experimental animals were fasted for 12 hours but allowed free access to water. Blood was collected from the orbital rim, and fasting insulin (FINS) in rats was measured using an ELISA insulin kit. The experimental procedure was performed according to the kit instructions. The insulin resistance index (IRI = FBG × FINS / 22.5), insulin sensitivity index (=ln〔1 / (FBG × FINS)〕), and insulin secretion index (HOMA-IS) were calculated using Turner's HOMA homeostasis model: 20 × fasting plasma insulin / (fasting blood glucose - 3.5);

[0101] 2.5. After the last administration, experimental animals were fasted for 12 hours but allowed free access to water. Blood was collected from the femoral artery and centrifuged at 3000 rpm for 15 minutes using a high-speed refrigerated centrifuge. The supernatant serum was then collected and stored at -20°C. ELISA kits for total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were used, and the experimental procedures were performed according to the kit instructions.

[0102] 2.6. After the last administration, the animals were fasted for 12 hours but allowed free access to water. Blood was collected from the femoral artery and centrifuged at 3000 rpm for 15 minutes using a high-speed refrigerated centrifuge. The supernatant serum was then collected and stored at -20°C. ELISA kits for superoxide dismutase (SOD) and malondialdehyde (MDA) were used, and the experimental procedures were performed according to the kit instructions.

[0103] 2.7. All data from the above experiments are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 16.0 software, and t-tests were used for inter-group comparisons.

[0104] 3. Experimental Results

[0105] (1) Observation under normal circumstances

[0106] During the drug administration period, rats in the blank control group showed normal physique, alertness, good mental state, pure white and glossy fur, and formed stools. Rats in the diabetic model group exhibited reduced activity, lethargy, dull and yellowish fur, and loose stools. Their food intake, water intake, and urine output significantly increased compared to the blank control group, and they gradually became emaciated, exhibiting pronounced symptoms of polyuria, polydipsia, polyphagia, and weight loss. Rats in the lycopene monomer group, saponin monomer group, and metformin hydrochloride positive drug group showed significant improvements in mental state and fur color, and their symptoms of polyuria, polydipsia, polyphagia, and weight loss gradually lessened. Mortality occurred in all groups. The model group had the highest mortality rate (8 rats), while the other groups had 4-6 rats each. At the end of the drug administration period, each group had more than 10 rats remaining.

[0107] (2) Food intake and water intake

[0108] The statistical results of the average food intake and water consumption of rats in each treatment group are shown in Tables 3-4.

[0109] Table 3. Average food intake of STZ-induced diabetic rats under different treatments.

[0110]

[0111] Note: Compared with the blank control group, ΔP < 0.01, which is highly significant; compared with the diabetes model group, **P < 0.01 is highly significant, and *P < 0.05 is significant, and the same applies below.

[0112] As shown in Table 3, compared with the blank control group, the rats in the model group had a significantly increased food intake (P<0.01), and the "polyphagia" symptoms caused by diabetes were obvious. Compared with the diabetes model group, the food intake of the barley glycoside monomer group and the saponin glycoside monomer group was significantly reduced after 7 days of administration (P<0.05), and the effect was comparable to that of the positive control drug, indicating that barley glycoside monomer and saponin glycoside monomer can improve the "polyphagia" symptoms caused by diabetes.

[0113] Table 4. Water intake in STZ-induced diabetic rats under different treatments

[0114]

[0115]

[0116] As shown in Table 4, compared with the blank control group, the water intake of the diabetic model group was significantly increased (P<0.01), and the "polydipsia" symptom caused by diabetes was obvious. Compared with the diabetic model group, the water intake of the phytoflavin monomer group and the saponin monomer group was significantly reduced after 14 days of administration (P<0.01), with effects comparable to the positive control group, indicating that phytoflavin monomer and saponin monomer can improve the "polydipsia" symptom caused by diabetes.

[0117] (3) Urine output

[0118] The statistical results of the average urine output of rats in each treatment group are shown in Table 5.

[0119] Table 5. Average urine output in STZ-induced diabetic rats under different treatments.

[0120]

[0121] As shown in Table 5, compared with the blank control group, the average daily urine output of the diabetic model rats was significantly increased (P<0.01), and the "polyuria" symptom caused by diabetes was obvious. The metformin hydrochloride group (positive control group) could reduce the urine output of STZ-induced diabetic rats after 14 days of administration (P<0.05). The phytoflavin monomer group and saponin monomer group could reduce the urine output of STZ-induced diabetic rats after 7 days of administration (P<0.05). This indicates that phytoflavin monomer and saponin monomer can improve the "polyuria" symptom caused by diabetes, and the effect is better than that of the positive control.

[0122] (4)Weight

[0123] The statistical results of the average urine output of rats in each treatment group are shown in Table 6.

[0124] Table 6 Body weight of STZ-induced diabetic rats under different treatments

[0125]

[0126] As shown in Table 6, the weight of rats in the blank control group continuously increased, while the weight of rats in the diabetic model group continuously decreased over time (P<0.01), indicating a significant weight loss symptom caused by diabetes. Compared with the diabetic model group, the weight of rats in the metformin hydrochloride, lycopene monomer, and saponin monomer groups increased over time after administration. Specifically, the weight of the saponin monomer group was significantly higher than that of the model group after 14 days of administration (P<0.05), and the weight of the lycopene monomer group was significantly higher than that of the model group after 21 days of administration (P<0.05). This indicates that lycopene monomer and saponin monomer can improve the weight loss symptoms caused by diabetes.

[0127] (5) Blood glucose

[0128] The statistical results of blood glucose levels and hypoglycemic effects in rats of each treatment group are shown in Table 7.

[0129] Table 7. Blood glucose levels and hypoglycemic effects of different STZ treatments in diabetic rats

[0130]

[0131] As shown in Table 7, compared with the blank control group, the fasting blood glucose level in the diabetes model group was significantly increased (P<0.001); compared with the diabetes model group, after 7 days of administration, the fasting blood glucose level in the positive drug metformin hydrochloride group was significantly decreased (P<0.05); after 14 days of administration, the fasting blood glucose levels in both the phytoflavin monomer group and the saponin monomer group were significantly decreased (P<0.05); after 28 days of treatment with the positive drug metformin hydrochloride, phytoflavin monomer, and saponin monomer, the blood glucose reduction rates were 59.77%, 48.22%, and 54.92%, respectively, indicating that both phytoflavin monomer and saponin monomer can significantly reduce fasting blood glucose.

[0132] As can be seen from Tables 3-7, the barley flavonoid monomer and saponin monomer provided by this invention can significantly improve the symptoms of "three highs and one low" in diabetic rats, and at the same time reduce the fasting blood glucose level in STZ-induced diabetic rats, indicating that barley flavonoid monomer and saponin monomer have a certain hypoglycemic effect.

[0133] (6) Postprandial blood glucose level

[0134] The results of postprandial blood glucose levels in rats in each treatment group are shown in Table 8.

[0135] Table 8. Effects of different treatments on glucose tolerance in STZ-induced diabetic rats

[0136]

[0137] As shown in Table 8, in the blank control group, blood glucose levels in rats increased significantly 0.5 hours after oral glucose administration, began to decrease after 0.5 hours, and returned to normal levels at 2 hours. In the model group, blood glucose levels increased sharply 0.5 hours after oral glucose administration, decreased somewhat at 1 hour, but at a slower rate, and remained higher than pre-oral glucose levels at 2 hours, indicating impaired glucose tolerance. Compared with the diabetic model group, the blood glucose increases in each treatment group were lower at 0.5 hours than in the model group, and the rate of decrease at 1 hour was significantly faster. After 2 hours, the blood glucose levels in the saponin monomer group were lower than pre-oral glucose levels, and the area under the blood glucose curve in the saponin monomer group was significantly lower than that in the positive control group. This indicates that both phytoglycoside monomer and saponin monomer can significantly reduce postprandial blood glucose levels in STZ-induced type II diabetic mice, and the saponin monomer has a greater effect on improving glucose tolerance in diabetic mice than the positive control drug metformin hydrochloride.

[0138] (7) Insulin

[0139] The insulin detection results of rats in each treatment group are shown in Table 9.

[0140] Table 9. Effects of different treatments on FINS, IRI, and ISI in STZ-induced diabetic rats.

[0141]

[0142] As shown in Table 9, compared with the blank control group, the fasting serum insulin (FINS) level and insulin resistance index (IRI) of the model group rats were significantly increased (P<0.001), while the insulin sensitivity index (ISI) and islet secretion index (HOMA-IS) were significantly decreased (P<0.001). Compared with the diabetic model group, each treatment group did not significantly change the insulin secretion index, but all of them could reduce the fasting serum insulin level and insulin resistance index of diabetic rats and increase the insulin sensitivity index. The effect of saponin monomer was better than that of metformin. This indicates that saponin monomer and saponin monomer can effectively improve the hyperinsulinemia and insulin resistance status induced by STZ in type II diabetic mice and increase the insulin sensitivity index, but they do not promote insulin secretion.

[0143] (8) Lipid metabolism in rats

[0144] The results of lipid metabolism detection in rats in each treatment group are shown in Table 10.

[0145] Table 10 Effects of different treatments on blood lipid levels in STZ-induced diabetic rats

[0146]

[0147] As shown in Table 10, after 28 days of drug treatment, compared with the diabetic model group, the serum TC, TG, and LDL levels in the metformin hydrochloride positive drug group, the phytoflavin monomer group, and the saponin monomer group were all significantly decreased (P<0.05), while HDL levels were all significantly increased (P<0.05). This indicates that phytoflavin monomer and saponin monomer can reduce the levels of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein (LDL-C) in the body, and increase the level of high-density lipoprotein (HDL-C), thereby improving lipid metabolism disorders and lowering blood glucose.

[0148] (9) Serum SOD and MDA

[0149] The results of serum SOD and MDA levels in rats in each treatment group are shown in Table 11.

[0150] Table 11 Effects of different treatments on serum SOD and MDA in STZ-induced diabetic rats

[0151]

[0152] As shown in Table 11, after 28 days of drug treatment, the fasting serum SOD activity in the metformin hydrochloride positive drug group, the phytoflavin monomer group, and the saponin monomer group was significantly higher than that in the diabetic model group (P<0.01). Compared with the diabetic model group, the fasting serum MDA content in the metformin hydrochloride positive drug group and the saponin monomer group was significantly lower (P<0.01), while the fasting serum MDA content in the phytoflavin monomer group decreased significantly (P<0.05). This indicates that phytoflavin monomer and saponin monomer can improve the body's antioxidant capacity to a certain extent, reduce oxidative damage caused by diabetes, and effectively control the occurrence of diabetic complications.

[0153] Test Example 2

[0154] Effects of barley flavin monomer and saponin monomer on glucose uptake by 3T3-L1 adipocytes

[0155] 1. Constructing a 3T3-L1 preadipocyte insulin resistance model

[0156] 3T3-L1 cells were fed at a dose of 1×10 4 Cells were seeded per well in 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator. Once the cells reached near confluence, differentiation was induced. Control group cells were cultured normally, while model group cells were induced in DMEM high-glucose medium containing 1 μM dexamethasone, 0.5 mM IBMX, and 10 μg / mL insulin (10% fetal bovine serum). After 48 hours, the medium was replaced with DMEM high-glucose medium containing 10 μg / mL insulin (10% fetal bovine serum) for further induction, followed by another 48 hours. After 12 days of continuous induction, oil red O staining and microscopic observation showed that over 90% of 3T3-L1 cells had differentiated into adipocytes, indicating mature differentiation. Cells were then cultured in DMEM high-glucose medium containing 1 μM dexamethasone for 48 hours. Glucose levels in the cell supernatant were measured using a glucose assay kit, and glucose consumption was calculated for each group. A significantly lower glucose consumption in the model group compared to the control group confirmed the successful establishment of the insulin-resistant cell model.

[0157] 2. Experimental Methods

[0158] After establishing the insulin resistance cell model, the model group was randomly divided into four groups: berberine monomer group, saponin monomer group, berberine group, and insulin resistance model group, for use in glucose consumption experiments.

[0159] Barley glycoside monomer group: The barley glycoside monomer obtained in Example 1 was prepared into a stock solution using DMSO, dissolved in DMEM high glucose medium, the old culture medium in the cells was discarded, and DMEM high glucose medium containing the sample to be tested was added, wherein the effective concentration of barley glycoside monomer was 34.4 μM.

[0160] Saponin monomer group: The saponin monomer obtained in Example 3 was prepared into a stock solution using DMSO, dissolved in DMEM high glucose medium, the old culture medium in the cells was discarded, and DMEM high glucose medium containing the sample to be tested was added, wherein the concentration of saponin monomer was 26.9 μM.

[0161] Berberine group: The old culture medium in the cells was discarded, and DMEM high glucose medium containing berberine was added, with the effective concentration of berberine being 10.0 μM;

[0162] The insulin resistance model group and the blank control group were respectively given culture medium containing DMSO;

[0163] After culturing for 24 hours, the culture medium of each treatment group was collected, the supernatant was taken, and the glucose content was determined by a glucose assay kit. The glucose consumption rate was calculated according to the following formula. All data are expressed as mean ± standard deviation. Data statistical analysis was performed using SPSS 16.0 software, and the t-test was used for inter-group comparisons. The results are shown in Table 12.

[0164] Glucose consumption rate (%) = [(initial glucose concentration - experimental well glucose concentration) / initial glucose concentration] × 100%.

[0165] Table 12 Effects of different treatments on glucose uptake by 3T3-L1 adipocytes

[0166]

[0167] Note: Compared with the control group, ΔP < 0.01, which is highly significant; compared with the insulin resistance model group, *P < 0.05, which is significant.

[0168] As shown in Table 12, compared with the control group, the glucose uptake capacity of 3T3-L1 adipocytes in the model group was significantly reduced, indicating insulin resistance. Compared with the diabetic model group, the positive control group (berberine, lycopene monomer, and saponin monomer) significantly improved the glucose uptake capacity of 3T3-L1 adipocytes (P<0.01). This indicates that both lycopene monomer and saponin monomer can promote glucose uptake by 3T3-L1 adipocytes, enhance cellular glucose utilization, and thus achieve a hypoglycemic effect.

[0169] Test Example 3

[0170] Determination of the inhibitory activities and inhibition types of α-glucosidase by barley flavin monomers and saponin monomers.

[0171] 1. Solution preparation

[0172] Preparation of PBS buffer: Solution A: 7.16 g disodium hydrogen phosphate (Na2HPO4·12H2O) dissolved in 100 mL double-distilled water; Solution B: 3.12 g sodium dihydrogen phosphate (NaH2PO4·2H2O) dissolved in 100 mL double-distilled water. Mix solutions A and B at a ratio of 49:51. Measure the pH using a pH meter; pH = 6.8, concentration 0.2 mol / L. Dilute to 0.05 mol / L before use. Sterilize with a 0.20 μm filter and store at -4℃ for later use. Preparation of substrate 4-nitrophenyl-α-D-glucopyranoside (PNPG): Prepare PNPG with diluted PBS to a concentration of 8.92 × 10⁻⁶. -3 mol / L, sterilized with a 0.20 μm filter membrane, aliquoted into 1.5 mL EP tubes, and frozen at -20℃ for later use; Preparation of α-glucosidase: Prepare a 1 U / mL storage solution of α-glucosidase with diluted PBS, sterilize with a 0.20 μm filter membrane, aliquot into 1.5 mL EP tubes, and freeze at -20℃ for later use. Dilute to 0.08 U / mL before use; Preparation of acarbose solution: Prepare a 12 mg / mL storage solution of acarbose with diluted PBS. Dilute before use.

[0173] Preparation of sample of phytoflavin monomer to be tested: Take 80 mg of phytoflavin monomer obtained in Example 1, dissolve it in a small amount of DMSO, and then prepare a preservation solution with a concentration of 80 mg / mL with PBS. Dilute to a gradient concentration before use.

[0174] Preparation of saponin monomer test sample: Take 80 mg of saponin monomer obtained in Example 3, dissolve it in a small amount of DMSO, and then prepare a preservation solution with a concentration of 80 mg / mL using PBS. Dilute to a gradient concentration before use.

[0175] 2. Establishment of the standard curve

[0176] Under inhibitor-free conditions, the reaction was carried out using a fully automated microplate reader in non-removable 96-well plates. The reaction mixture was 200 μL: 125 μL of 0.05 mol / L PBS and 8.92 × 10⁻⁶ μL of PBS. -3 25 μL of α-glucosidase (AG) was added to 25 μL of PNPG (mol / L), and the solution was incubated at 37℃ for 30 min. The absorbance was measured at 405 nm. The concentration gradient of AG was 0, 0.04, 0.06, 0.08, 0.10, and 0.12 U / mL. A graph was plotted based on the corresponding OD values ​​at different enzyme concentrations. The results are shown below. Figure 7As shown, the equation of the curve is Y = 6.4321X - 0.0113, R 2 =0.9985, indicating a good linear relationship between α-glucosidase concentrations in the range of 0–0.12 U / mL.

[0177] 3. Enzyme inhibitory activity assay

[0178] The reaction was performed using a fully automated microplate reader in a non-removable 96-well plate. The reaction mixture was 200 μL: 125 μL of 0.05 mol / L PBS and 8.92 × 10⁻⁶ μL of PBS. -3 Add 25 μL of the test sample and 25 μL of 0.08 U / mL AG to 25 μL of PNPG (mol / L). Incubate at 37℃ for 30 min, and measure the absorbance at 405 nm. Use PBS instead of enzyme solution as a blank control, and acarbose as a positive control. Perform three parallel experiments for each sample at the same concentration, and take the average value. Substitute the average value into the standard curve in step 2 to obtain enzyme activity, and calculate the inhibition rate and IC50 using the following formula. 50 The values ​​are shown in Table 13.

[0179] Wherein, A1: original enzyme activity; A2: enzyme activity after adding inhibitor; A3: blank control; A4: sample background;

[0180] Table 13 Inhibitory effects of barley glycoside monomers and saponin monomers on α-glucosidase

[0181]

[0182]

[0183] As shown in Table 13, both phytoflavin monomer and saponin monomer exhibit α-glucosidase inhibitory activity. The IC50 of the positive control drug acarbose is 4.41 mg / mL, the IC50 of phytoflavin monomer is 6.64 mg / mL, and the IC50 of saponin monomer is 2.75 mg / mL. Saponin monomer shows superior α-glucosidase inhibitory activity compared to the positive control drug acarbose.

[0184] 4. Determination of α-glucosidase inhibition type

[0185] The α-glucosidase inhibition type was determined using a chemical kinetic method, with the slope indicating the inhibition type. Reversible inhibitors yielded a set of straight lines passing through the origin but with different slopes, while irreversible inhibitors yielded a set of parallel lines not passing through the origin but with the same slope as the control. Specifically, different amounts of α-glucosidase and the test sample were added to the reaction system, and linear relationships were plotted between the reaction rate and the enzyme amount for different test samples, generating EV graphs. The α-glucosidase amounts were 0.08 U, 0.10 U, 0.12 U, 0.14 U, and 0.16 U, and the test samples were phytoxicin monomer or saponin monomer at concentrations of 0, 5 mg / mL, and 10 mg / mL. The results showed that both phytoxicin monomer and saponin monomer exhibited irreversible inhibition of the enzyme. Figure 8 and Figure 9 ).

[0186] Example 5

[0187] A type of blood sugar-lowering solid beverage food

[0188] In Example 1, 20% maltodextrin, 0.3% sodium carboxymethyl cellulose, and 5% sucrose or xylitol were added to the barley flavonoid monomer, and the mixture was stirred using a SYH laboratory three-dimensional motion mixer. The mixture was then granulated using conventional methods to obtain a solid beverage.

[0189] Example 6

[0190] A type of blood sugar-lowering solid beverage food

[0191] In Example 1, 20% maltodextrin, 0.3% sodium carboxymethyl cellulose, and 5% sucrose or xylitol were added to the saponin monomer prepared in the laboratory. The mixture was then mixed using a SYH laboratory three-dimensional motion mixer and granulated using conventional methods to obtain a solid beverage.

[0192] Example 7

[0193] A type of blood sugar-lowering compressed candy health food

[0194] In Example 2, 20% maltitol or xylitol or aspartame or stevioside, 15% microcrystalline cellulose and 5% magnesium stearate were added to the barley flavonoid monomer and saponin monomer prepared. The mixture was then mixed using a SYH laboratory three-dimensional motion mixer, dry granulated, and then tableted using a ten-punch tablet press (ZPSX) (pressure 40-60KN) to obtain compressed candy.

[0195] Example 8

[0196] A capsule medicine for treating diabetes

[0197] In Example 3, 30-50% lactose or pregelatinized starch, 2% magnesium stearate, and 5% talc or micronized silica gel were added to the saponin monomer. The mixture was then mixed using a SYH laboratory three-dimensional motion mixer, dry granulated, and then filled into capsules using an NJP-2-200C fully automatic capsule filling machine.

[0198] Example 9

[0199] A microcapsule drug for treating diabetes

[0200] The phytoflavin monomer prepared in Example 3 was mixed with sodium alginate at a ratio of 1:2 g / g and uniformly dispersed in water to achieve a sodium alginate concentration of 2%, resulting in solution A of daisy leaf gentianone and sodium alginate. A chitosan acetic acid solution with a chitosan mass concentration of 1.0% was prepared, and a certain amount of CaCl2 solution was added to achieve a mass concentration of 1.8%. The pH was adjusted to 5.5 with an alkaline solution of a certain concentration, and the mixture was stirred evenly to obtain solution B. Solution A was sprayed into solution B with particles of 150 μm diameter using a microcapsule granulator, while stirring to carry out a film-forming reaction. The film-forming reaction time was 20 minutes. The microspheres were collected, washed, and freeze-dried to obtain microcapsules.

[0201] Example 10

[0202] A type of drop pill medicine for treating diabetes

[0203] In Example 4, 55% pharmaceutical matrix PEG-3350 and 40% pharmaceutical matrix PEG-8000 were added to the barley flavin monomer and saponin monomer prepared. After complete melting in an 85°C water bath, the mixture was stirred and poured into the loading tank of a small pelleting machine. After stirring and keeping warm for 30 minutes, the pellets were dropped into dimethyl silicone oil coolant at a dropping distance of 10 cm and a rate of 30 drops / min. After the pellets completely shrank and solidified, they were removed and the coolant on the surface of the pellets was removed using an oil slinger to obtain the pellets.

[0204] As can be seen from the above, this invention can separate and purify high-purity phytoflavin monomers and saponin monomers, which have significant hypoglycemic effects and can be used to prepare products for lowering blood sugar, preventing and / or treating diabetes. Furthermore, the separation and purification method provided by this invention is simple to operate, easy to implement for industrial production, and exhibits good repeatability and stability.

[0205] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for separating and purifying phytoflavin and saponin monomers, characterized in that, Includes the following steps: (1) Mix the raw materials containing phytoflavin and saponin with solvent, heat and reflux to extract, concentrate the extract to obtain crude extract; The solvent is water or an aqueous ethanol solution; the volume concentration of ethanol in the aqueous ethanol solution is >0 and ≤40%; when the solvent is an aqueous ethanol solution, the ethanol in the extract is separated and then concentrated to obtain a crude extract; (2) After dissolving the crude extract in hot water at 70°C, it was adsorbed onto AB-8 macroporous resin. After complete adsorption, it was eluted with water until colorless, and then eluted with an ethanol aqueous solution with a volume concentration of 10-50%. The ethanol eluent was collected, concentrated, and dried to obtain the total flavonoid extract. (3) After dissolving the total flavonoid extract in an ethanol aqueous solution with a volume concentration of 10-40%, the extract was separated by medium-pressure preparative chromatography. Under the detection condition of 340 nm wavelength, the eluents of barley flavonoid monomer and saponin flavonoid monomer were collected according to the chromatogram. The separation using medium-pressure preparative chromatography includes isocratic elution using an ethanol-water solution with a volume concentration of 10-30% as the mobile phase. (4) Separate the ethanol from the eluent of the barley flavonoid monomer and the eluent of the saponin monomer, and dry them to obtain barley flavonoid monomer and saponin monomer respectively. The raw materials containing phytoflavin and saponin are barley seedlings and / or barley seedlings. The elution with water is performed by eluting with 1-5 column volumes of water at a flow rate of 100-200 L / h. The elution with an ethanol aqueous solution of 10-50% by volume is performed by eluting with 3-6 column volumes of an ethanol aqueous solution of 10-50% by volume; the flow rate of the ethanol aqueous solution is 100-200 L / h.

2. The separation and purification method according to claim 1, characterized in that, The heating and reflux extraction is performed 1 to 3 times, with each heating and reflux extraction lasting 1 to 3 hours; the heating and reflux extraction temperature is above 90°C.

3. The separation and purification method according to claim 2, characterized in that, During each heating and reflux extraction, the ratio of the raw material containing phytoflavin and saponin to the ethanol aqueous solution is 1g:20~30mL.

4. The separation and purification method according to claim 1, characterized in that, The loading rate of the AB-8 macroporous resin is 50-200 L / h; The adsorption time is ≥24h.

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