Separation process of novel-structure alpha-glucosidase inhibitor Lycbarbarspermides Q in red wolfberry and application of novel-structure alpha-glucosidase inhibitor Lycbarbarspermides Q

Abstract

The invention discloses a separation process of an alpha-glucosidase inhibitor Lycbarbarspermides Q with a new structure in red Chinese wolfberry fruits and application of the alpha-glucosidase inhibitor Lycbarbarspermides Q with the new structure in the red Chinese wolfberry fruits. The specific preparation process comprises the following seven steps: extraction, on-line alpha-glucosidase inhibitor component identification, hydrophilic water pressure preparation column coarse separation, reversed-phase high-pressure preparation column preparation, hydrophilic high-pressure preparation column preparation, gel filtration chromatography and in-vitro alpha-glucosidase inhibition experiment. The method is low in cost, and the product purity is greater than 95%; the technical means adopted by the invention can be used for large-scale production: the raw material requirement is not high, the cost is low, and batch material preparation is easy; in the preparation process, solvents and separation materials used by an extraction solvent, a hydrophilic preparative column, a reversed-phase preparative column, gel chromatography and a reversed-phase chromatographic column can be recycled.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of separation of alpha-glucosidase inhibitors, and particularly relates to a separation process of a new structure alpha-glucosidase inhibitor Lycibarbarspermidines Q in Lycium barbarum and application thereof. BACKGROUND

[0002] Lycium barbarum L. is a plant of Solanaceae Lycium, mainly distributed in northwest China, and has a long history of medicinal and edible use. The fruit is rich in various bioactive components, such as polysaccharides, carotenoids, flavonoids, vitamins and minerals. Lycium barbarum is widely used in traditional Chinese medicine for nourishing liver and kidney, improving eyesight, anti-aging and enhancing immunity. Modern studies have shown that Lycium barbarum is rich in Lycium barbarum polysaccharides, flavonoids, carotenoids, phenolic acids and alkaloids, and has anti-inflammatory, antioxidant, hypoglycemic, immunomodulatory, neuroprotective and other pharmacological activities, and has significant development potential. Literature reports that phenylpropanoids and flavonoids (caffeic acid, quercetin-3-O-rutin glycoside and kaempferol-3-O-rutin glycoside) in Lycium barbarum have antioxidant effects. Seven different types of polyphenolic compounds in Lycium barbarum, including phenylpropanoids, coumarins, lignans, flavonoids, isoflavones, chlorogenic acids, p-hydroxybenzaldehyde and p-hydroxybenzoic acid compounds, have antioxidant and free radical scavenging abilities.

[0003] As a natural product with rich bioactive components, it is necessary to further accelerate the quality evaluation, production and sales of Lycium barbarum and the research and development pace of related new drugs. SUMMARY

[0004] Based on the above technical problems, the purpose of the present application is to provide a separation process of a new structure alpha-glucosidase inhibitor Lycibarbarspermidines Q in Lycium barbarum and application thereof.

[0005] The present application protects a separation process of a new structure alpha-glucosidase inhibitor Lycibarbarspermidines Q in Lycium barbarum, which specifically comprises the following steps: Step 1, extraction: Lycium barbarum is dried in the shade, coarsely crushed, and then extracted with 100% ethanol at a solid-liquid ratio of 1 g: 10-20 mL. The extraction is performed under vacuum heating reflux for 2-4 times, each time for 2-4 h. The filtrate is filtered, combined, and then concentrated under vacuum and reduced pressure to obtain concentrated solution A; Step 2, online α-glucosidase inhibitor component identification: add methanol with a mass of 5-10 times of the total sample of Lycium ruthenicum Gal to dissolve, the volume concentration of the methanol is 70%-90%, prepare the sample concentration to be 50.0-100.0 mg / mL, filter through a 0.45 μm microporous filter membrane to obtain a Lycium ruthenicum Gal methanol sample solution, i.e. filtrate B; take 1 mL of the filtrate B, and use an online HPLC-FLD chromatography system to identify that the Lycium ruthenicum Gal contains the target component α-glucosidase inhibitor; wherein the online HPLC-FLD chromatography system, the first part is a high performance liquid chromatograph, an Odyssil C18 (250x4.6 mm, 5 μm) chromatographic column is used, and the detection wavelength is 300 nm; the second part is a fluorescence detector and a high performance liquid chromatograph pump, the excitation wavelength and the emission wavelength of the fluorescence detector are 219 nm and 332 nm respectively, and the high performance liquid chromatograph pump pumps in an α-glucosidase aqueous solution; Step 3, hydrophilic medium pressure preparation column crude separation: wet sample the filtrate B and inject into a medium pressure chromatographic column filled with hydrophilic filler, use the chromatographic column to separate the sample, use a wavelength of 300 nm ultraviolet detector to monitor the effluent during the separation process, according to the detection results, collect the target chromatographic peak component in the preparation chromatogram, through high performance liquid chromatography HPLC analysis, compare the retention time of the component Fr6 with the total sample of Lycium ruthenicum Gal, confirm the active target component, and after vacuum and reduced pressure drying treatment, the target component Fr6 is obtained; Step 4, reverse phase high pressure preparation column preparation: the component Fr6 is separated by a reverse phase chromatographic column, detected by a wavelength of 300 nm ultraviolet detector, and the corresponding chromatographic peak component Fr6-3 in the preparation chromatogram is collected, through high performance liquid chromatography HPLC analysis, compare the retention time of the component Fr6-3 with the component Fr6, confirm the active target component, and vacuum and reduced pressure drying obtains the target component Fr6-3; Step 5, hydrophilic high pressure preparation column preparation: the component Fr6-3 is separated by a hydrophilic chromatographic column, detected by a wavelength of 300 nm ultraviolet detector, and the corresponding chromatographic peak component in the preparation chromatogram is collected, through high performance liquid chromatography HPLC analysis, compare the retention time of the component Fr6-3-1 with the component Fr6-3, confirm the active target component, and vacuum and reduced pressure drying obtains the target component Fr6-3-1; Step 6, Gel Filtration Chromatography: The component Fr6-3-1 was dissolved in methanol solution to prepare a sample concentration of 100.0 mg / mL. The solution was filtered through a 0.45 μm microporous membrane to obtain filtrate C. Filtrate C was then subjected to gel chromatography for desalting, acid removal, and removal of small molecule impurities. The sample was detected using a 300 nm UV detector. The main chromatographic peak components in the preparative chromatogram were collected. Each component was dried under vacuum to obtain the target new compound with a purity greater than 95%, named LycibarbarspermidinesQ, with the following chemical structural formula: ; Step 7, In vitro α-glucosidase inhibition experiment: Add 10 μL of the target compound sample solution diluted with PBS buffer, 20 μL of α-glucosidase solution prepared with 0.2 U / mL PBS buffer, 10 μL of pNPG solution prepared with 3.125 mM distilled water, and 70 μL of PBS buffer to a microplate sequentially, so that the final reaction concentrations of the samples are 0.25, 0.50, 1.00, 2.00, 3.00, and 4.00 mM, respectively. Incubate at 37℃ for 15 min, add 80 μL of NaCO3 solution prepared with 0.2 M distilled water to terminate the reaction, incubate at 37℃ for 15 min, and measure the absorbance at 405 nm. Acarbose was used as a positive control. The blank group used an equal volume of PBS buffer instead of the sample solution, the blank control group used an equal volume of PBS buffer instead of the sample and enzyme solution, and the sample control group used an equal volume of PBS buffer instead of the enzyme solution. The inhibition rate of the samples against α-glucosidase is as follows: In the formula, ODs, ODs0, and OD b OD b0 These represent the absorbance values ​​of the sample group, sample control group, blank group, and blank control group, respectively.

[0006] Furthermore, in steps 1, 3, 4, 5, and 6, the conditions for vacuum concentration are: vacuum degree 50~250 mbar and temperature 40~60℃.

[0007] Further, in step 2, the mobile phase A of the high-performance liquid chromatograph is a 0.2% trifluoroacetic acid-water solution, and the mobile phase B is an acetonitrile solution, with a flow rate of 1.0 mL / min for 0~30 min, 5%-30% B; the concentration of α-glucosidase pumped in by the high-performance liquid chromatograph pump is 15 μg / mL, and the flow rate of the mobile phase is 1 mL / min.

[0008] Further, in step 3, the working parameters of the hydrophilic high-pressure preparation column used for the separation of the components are as follows: the column length of the preparation column is 460 mm, the column diameter is 49 mm, the stationary phase is Click XION, the mobile phase A is 0.2% trifluoroacetic acid, the mobile phase B is acetonitrile, the chromatographic conditions are 100%-70% B in 0-60 min, the injection volume is 50 g, and the flow rate is 80 mL / min; high-performance liquid chromatography analysis comparison is performed using an Odyssil C18 (250x4.6 mm, 5 μm) chromatographic column, the detection wavelength is 300 nm, the mobile phase A used is 0.2% trifluoroacetic acid-water solution, the mobile phase B used is acetonitrile solution, and the flow rate of the mobile phase is 1.0 mL / min in accordance with 5%-30% B in 0-30 min.

[0009] Further, in step 4, the working parameters of the reverse-phase high-pressure preparation column used for the preparation of the component Fr6 are as follows: the column length of the preparation column is 250 mm, the column diameter is 20 mm, the stationary phase of the reverse-phase chromatographic column is 5 μm water-resistant C18, the mobile phase A is 0.2% trifluoroacetic acid-water solution, the mobile phase B is methanol solution, elution is performed in accordance with 10%-35% B in 0-70 min, the injection volume is 4 mL, and the flow rate is 19 mL / min; high-performance liquid chromatography analysis comparison is performed using an Odyssil C18 (250x4.6 mm, 5 μm) chromatographic column, the detection wavelength is 300 nm, the mobile phase A used is 0.2% trifluoroacetic acid-water solution, the mobile phase B used is acetonitrile solution, and the flow rate of the mobile phase is 1.0 mL / min in accordance with 5%-30% B in 0-30 min.

[0010] Further, in step 5, the working parameters of the hydrophilic high-pressure preparation column used for the preparation of the Fr6-3 component are as follows: the column length of the preparation column is 250 mm, the column diameter is 20 mm, the stationary phase of the hydrophilic chromatographic column is 5 μm HILIC-D hydrophilic filler, the mobile phase A is 0.2% trifluoroacetic acid-water solution, the mobile phase B is acetonitrile solution, elution is performed in accordance with 82% B in 0-60 min, the injection volume is 4 mL, and the flow rate is 19 mL / min; high-performance liquid chromatography analysis comparison is performed using an Odyssil C18 (250x4.6 mm, 5 μm) chromatographic column, the detection wavelength is 300 nm, the mobile phase A used is 0.2% trifluoroacetic acid-water solution, the mobile phase B used is acetonitrile solution, and the flow rate of the mobile phase is 1.0 mL / min in accordance with 5%-30% B in 0-30 min.

[0011] Further, in step 6, the working parameters of the gel chromatographic column are as follows: the column length of the chromatographic column is 1200 mm, the column diameter is 30 mm, the stationary phase of the gel chromatographic column is HW-40C, the mobile phase B is methanol, and the flow rate is 1 mL / min in accordance with 100% B in 0-1200 min.

[0012] The application also provides application of the new alpha-glucosidase inhibitor Lycibarbarispermidines Q separated from the above-mentioned Lycium barbarum in preparation of a blood sugar reducing medicine or health food, wherein the alpha-glucosidase inhibitor Lycibarbarispermidines Q is used as an effective component to be prepared into various medicinal preparations by a conventional method and in combination with any pharmaceutically acceptable carrier, or used as an effective component to be prepared into various health foods by a conventional method and in combination with any food scientifically acceptable carrier.

[0013] Compared with the prior art, the application has the following beneficial effects: (1) The application has low cost and high product purity The extraction solvent, the hydrophilic preparation column, the reverse phase preparation column, the gel chromatography, the solvent used in the reverse phase chromatography column and the separation material in the preparation process of the application can be recycled; the chromatographic separation material used can be reused; the recycled solvent and the reused separation material ensure that the average cost in the separation process is relatively low, and the high pressure chromatographic separation can ensure that the purity of the product is greater than 95%.

[0014] (2) The preparation method of the application can realize the need of large-scale production The raw material requirement is not high, the cost is low, and the wild or market sold Lycium barbarum can be used, which is easy to prepare in batches; ethanol extraction is easy to operate.

[0015] (3) The application utilizes the HPLC-FLD identification function component High performance liquid chromatography-fluorescence detector (HPLC-FLD) is an analysis method combining high performance liquid chromatography separation technology and enzyme solution constantly pumped into fluorescence detection; the application first applies this technology to the chromatographic identification field of compound target / protein function components, and thus, the applicant names this application in the laboratory as: online functional component chromatographic recognition system for target enzymes / proteins (Online functional components chromatographic recognition system for target enzymes / proteins), which is abbreviated as HPLC-FLD or UV-FLD. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is an online HPLC-FLD identification chromatogram of the component of the Lycium barbarum of the application containing the target component; Figure 2 It is a hydrophilic medium pressure preparation column separation chromatogram of the ethanol extract of the Lycium barbarum of the application; Figure 3 It is a component total sample and Fr6 comparison chromatogram of the application; Figure 4 Reversed phase high pressure preparative liquid chromatogram of component Fr6 of the present application; Figure 5 Comparative chromatogram of component Fr6 and Fr6-3 of the present application; Figure 6 Hydrophilic high pressure preparative liquid chromatogram of component Fr6-3-1 of the present application; Figure 7 Gel filtration chromatogram of component Fr6-3-1 of the present application; Figure 8 Purity and inhibitory activity on α-glucosidase verification chromatogram of new structure α-glucosidase inhibitor Lycibarbarspermidines Q of the present application; Figure 9 Half inhibitory concentration IC50 of new structure α-glucosidase inhibitor Lycibarbarspermidines Q of the present application; 50 ; Figure 10 High resolution mass spectrum of new structure α-glucosidase inhibitor Lycibarbarspermidines Q of the present application; Figure 11 H NMR nuclear magnetic resonance spectrum of new structure α-glucosidase inhibitor Lycibarbarspermidines Q of the present application; 1 Figure 12 C NMR nuclear magnetic resonance spectrum of new structure α-glucosidase inhibitor Lycibarbarspermidines Q of the present application; 13 Figure 13 HSQC spectrum of new structure α-glucosidase inhibitor Lycibarbarspermidines Q of the present application; Figure 14 HMBC spectrum of new structure α-glucosidase inhibitor Lycibarbarspermidines Q of the present application; Figure 15 H-HCOSY spectrum of new structure α-glucosidase inhibitor Lycibarbarspermidines Q of the present application; Figure 16 NOESY spectrum of new structure α-glucosidase inhibitor Lycibarbarspermidines Q of the present application; Figure 17 TOCSY spectrum of new structure α-glucosidase inhibitor Lycibarbarspermidines Q of the present application;​​ Figure 18 DEPT spectrum of the new structure α-glucosidase inhibitor Lycibarbarspermidines Q of the present application; Figure 19 The planar chemical structure formula diagram of the new structure α-glucosidase inhibitor Lycibarbarspermidines Q of the present application. DETAILED DESCRIPTION

[0017] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0018] Embodiment 1 A separation process of the new structure α-glucosidase inhibitor Lycibarbarspermidines Q in red wolfberry, specifically comprising the following steps: Step 1, extraction: 500 g of red wolfberry is dried in the shade, coarsely crushed, and then extracted with 100% ethanol at a material-liquid ratio of 1 g:10 mL. The extraction is performed under vacuum heating reflux for 4 times, 2 h each time. The filtrate is filtered, combined, and then concentrated under vacuum and reduced pressure to obtain concentrated liquid A. The concentration under vacuum and reduced pressure is performed at a vacuum degree of 50 mbar and a temperature of 40℃. Step 2, online α-glucosidase binding agent component identification: 5 times the mass of methanol is added to the total sample of red wolfberry for dissolution. The volume concentration of methanol is 90%, and the sample concentration is prepared to be 100.0 mg / mL. The sample solution is filtered through a 0.45 μm microporous filter to obtain a methanol sample solution of red wolfberry, i.e. filtrate B. 1 mL of the filtrate B is used to identify the α-glucosidase inhibitor containing the target component in red wolfberry by using an online HPLC-FLD chromatography system. The online HPLC-FLD chromatography system comprises a high performance liquid chromatograph as the first part, an Odyssil C18 (250×4.6 mm, 5 μm) chromatographic column, and a detection wavelength of 300 nm. The second part comprises a fluorescence detector and a high performance liquid chromatograph pump. The excitation wavelength and the emission wavelength of the fluorescence detector are 219 nm and 332 nm, respectively. The high performance liquid chromatograph pump pumps in an α-glucosidase aqueous solution with a concentration of 15 μg / mL at a flow rate of 1 mL / min. The mobile phase A used by the high performance liquid chromatograph is 0.2% trifluoroacetic acid-water solution, and the mobile phase B is acetonitrile solution. According to 0~30 min, 5%-30% B, the flow rate of the mobile phase is 1.0 mL / min. Step 3, column crude separation in hydrophilic medium pressure: the filtrate B is wet loaded into a medium pressure chromatographic column filled with hydrophilic packing, and the sample is separated by using the chromatographic column. In the separation process, an ultraviolet detector with a wavelength of 300 nm is used to monitor the effluent, and according to the detection results, the target chromatographic peak components in the preparative chromatogram are collected. The active target component is confirmed by comparing the retention time of component Fr6 with that of the total sample of red medlar. After vacuum and reduced pressure drying treatment, 2.08 g of target component Fr6 is obtained. The vacuum and reduced pressure concentration conditions are a vacuum degree of 50 mbar and a temperature of 40°C. The working parameters of the hydrophilic medium pressure preparative column separation are a column length of 460 mm, a diameter of 49 mm, a stationary phase of Click XION, a mobile phase A of 0.2% trifluoroacetic acid, a mobile phase B of acetonitrile, a chromatographic condition of 100%-70% B in 0-60 min, a sample injection amount of 50 g, and a flow rate of 80 mL / min. The high performance liquid chromatograph analysis comparison uses an Odyssil C18 (250x4.6 mm, 5 μm) chromatographic column, a detection wavelength of 300 nm, a mobile phase A of 0.2% trifluoroacetic acid-water solution, a mobile phase B of acetonitrile solution, and a flow rate of 1.0 mL / min in 5%-30% B in 0-30 min; Step 4, reverse phase high pressure preparative column preparation: component Fr6 is separated by a reverse phase chromatographic column, detected by an ultraviolet detector with a detection wavelength of 300 nm, and the corresponding chromatographic peak component Fr6-3 in the preparative chromatogram is collected. The active target component is confirmed by comparing the retention time of component Fr6-3 with that of component Fr6, and 85.63 mg of target component Fr6-3 is obtained by vacuum and reduced pressure drying. The vacuum and reduced pressure concentration conditions are a vacuum degree of 50 mbar and a temperature of 40°C. The working parameters of the reverse phase high pressure preparative column preparation of component Fr6 are a column length of 250 mm, a diameter of 20 mm, a reverse phase chromatographic column stationary phase of 5 μm water-resistant C18, a mobile phase A of 0.2% trifluoroacetic acid-water solution, a mobile phase B of methanol solution, elution in 10%-35% B in 0-70 min, a sample injection volume of 4 mL, and a flow rate of 19 mL / min. The high performance liquid chromatograph analysis comparison uses an Odyssil C18 (250x4.6 mm, 5 μm) chromatographic column, a detection wavelength of 300 nm, a mobile phase A of 0.2% trifluoroacetic acid-water solution, a mobile phase B of acetonitrile solution, and a flow rate of 1.0 mL / min in 5%-30% B in 0-30 min; Step 5, hydrophilic high-pressure preparative column preparation: the component Fr6-3 is separated by a hydrophilic chromatographic column, detected by a UV detector with a detection wavelength of 300 nm, and the corresponding chromatographic peak components in the preparative chromatogram are collected. The active target component is confirmed by comparing the retention time of component Fr6-3-1 with that of component Fr6-3 through high performance liquid chromatography (HPLC) analysis, and the target component Fr6-3-1 is obtained by vacuum concentration at a vacuum degree of 50 mbar and a temperature of 40°C. The working parameters of the hydrophilic high-pressure preparative column preparation of the Fr6-3 component are as follows: preparative column length 250 mm, diameter 20 mm, hydrophilic chromatographic column stationary phase 5 μm HILIC-D hydrophilic packing, mobile phase A 0.2% trifluoroacetic acid-water solution, mobile phase B acetonitrile solution, elution according to 0-60 min, 82% B, sample volume 4 mL, flow rate 19 mL / min; high performance liquid chromatography analysis comparison, using Odyssil C18 (250×4.6 mm, 5 μm) chromatographic column, detection wavelength 300 nm, using mobile phase A 0.2% trifluoroacetic acid-water solution, mobile phase B acetonitrile solution, according to 0-30 min, 5%-30% B, mobile phase flow rate 1.0 mL / min; Step 6, gel filtration chromatography: the component Fr6-3-1 is dissolved in a methanol solution to prepare a sample concentration of 100.0 mg / mL, filtered through a 0.45 μm microporous filter to obtain a filtrate C, and the filtrate C is desalted, deacidified and removed of small molecular impurities by a gel chromatographic column. The components are detected by a UV detector with a detection wavelength of 300 nm, and the main chromatographic peak components in the preparative chromatogram are collected. Each component is vacuum-concentrated at a vacuum degree of 50 mbar and a temperature of 40°C to obtain 13.05 mg of the target new compound with a purity of more than 95%, named Lycibarbarspermidines Q, with a chemical structural formula (see Figure 1 for details). Figure 19 Step 6, gel filtration chromatography: the component Fr6-3-1 is dissolved in a methanol solution to prepare a sample concentration of 100.0 mg / mL, filtered through a 0.45 μm microporous filter to obtain a filtrate C, and the filtrate C is desalted, deacidified and removed of small molecular impurities by a gel chromatographic column. The components are detected by a UV detector with a detection wavelength of 300 nm, and the main chromatographic peak components in the preparative chromatogram are collected. Each component is vacuum-concentrated at a vacuum degree of 50 mbar and a temperature of 40°C to obtain 13.05 mg of the target new compound with a purity of more than 95%, named Lycibarbarspermidines Q, with a chemical structural formula (see Figure 1 for details).

[0019] Example 2 A separation process of a new structural α-glucosidase inhibitor Lycibarbarspermidines Q in Lycium barbarum L. specifically includes the following steps: Step 1, extraction: 1000 g of dried Lycium barbarum L. was coarsely crushed and extracted with 100% ethanol at a material-to-liquid ratio of 1 g: 15 mL for 3 h each time by vacuum heating and reflux extraction for 3 times, filtered, the filtrate was combined, and the filtrate was concentrated under vacuum and reduced pressure to obtain concentrated liquid A, wherein the vacuum and reduced pressure concentration conditions were a vacuum degree of 150 mbar and a temperature of 50°C; Step 2, online α-glucosidase binding agent component identification: 8 times the mass of methanol was added to the Lycium barbarum L. total sample for dissolution, the volume concentration of methanol was 80%, the sample concentration was prepared to be 80.0 mg / mL, filtered through a 0.45 μm microporous filter to obtain a Lycium barbarum L. methanol sample solution, i.e., filtrate B; 1 mL of filtrate B was used to identify the α-glucosidase inhibitor containing target components in Lycium barbarum L. using an online HPLC-FLD chromatography system; wherein the online HPLC-FLD chromatography system, the first part was a high performance liquid chromatograph, an Odyssil C18 (250×4.6 mm, 5 μm) chromatographic column was used, and the detection wavelength was 300 nm; the second part was a fluorescence detector and a high performance liquid chromatograph pump, the excitation wavelength and the emission wavelength of the fluorescence detector were 219 nm and 332 nm, respectively, the high performance liquid chromatograph pump pumped α-glucosidase aqueous solution with a concentration of 15 μg / mL, and the flow rate of the mobile phase was 1 mL / min; wherein the high performance liquid chromatograph used a mobile phase A of 0.2% trifluoroacetic acid-water solution and a mobile phase B of acetonitrile solution, according to 0-30 min, 5%-30% B, the flow rate of the mobile phase was 1.0 mL / min; Step 3, column crude separation in hydrophilic medium pressure: the filtrate B is wet loaded into a medium pressure chromatographic column filled with hydrophilic packing, and the sample is separated by using the chromatographic column. In the separation process, an ultraviolet detector with a wavelength of 300 nm is used to monitor the effluent, and according to the detection results, the target chromatographic peak components in the preparative chromatogram are collected. The active target component is confirmed by comparing the retention time of component Fr6 with that of the total sample of red medlar. After vacuum and reduced pressure drying treatment, 5.33 g of target component Fr6 is obtained. The vacuum and reduced pressure concentration conditions are a vacuum degree of 150 mbar and a temperature of 50°C. The working parameters of the hydrophilic medium pressure preparative column separation are a column length of 460 mm, a diameter of 49 mm, a stationary phase of Click XION, a mobile phase A of 0.2% trifluoroacetic acid, a mobile phase B of acetonitrile, a chromatographic condition of 100%-70% B in 0-60 min, a sample injection amount of 50 g, and a flow rate of 80 mL / min. The high performance liquid chromatograph analysis comparison uses an Odyssil C18 (250x4.6 mm, 5 μm) chromatographic column, a detection wavelength of 300 nm, a mobile phase A of 0.2% trifluoroacetic acid-water solution, a mobile phase B of acetonitrile solution, and a flow rate of 1.0 mL / min in 5%-30% B in 0-30 min; Step 4, reverse phase high pressure preparative column preparation: component Fr6 is separated by a reverse phase chromatographic column, detected by an ultraviolet detector with a detection wavelength of 300 nm, and the corresponding chromatographic peak component Fr6-3 in the preparative chromatogram is collected. The active target component is confirmed by comparing the retention time of component Fr6-3 with that of component Fr6, and 165.33 mg of target component Fr6-3 is obtained by vacuum and reduced pressure drying. The vacuum and reduced pressure concentration conditions are a vacuum degree of 150 mbar and a temperature of 50°C. The working parameters of the reverse phase high pressure preparative column preparation of component Fr6 are a column length of 250 mm, a diameter of 20 mm, a reverse phase chromatographic column stationary phase of 5 μm water-resistant C18, a mobile phase A of 0.2% trifluoroacetic acid-water solution, a mobile phase B of methanol solution, elution in 10%-35% B in 0-70 min, a sample injection volume of 4 mL, and a flow rate of 19 mL / min. The high performance liquid chromatograph analysis comparison uses an Odyssil C18 (250x4.6 mm, 5 μm) chromatographic column, a detection wavelength of 300 nm, a mobile phase A of 0.2% trifluoroacetic acid-water solution, a mobile phase B of acetonitrile solution, and a flow rate of 1.0 mL / min in 5%-30% B in 0-30 min; Step 5, hydrophilic high-pressure preparative column preparation: the component Fr6-3 is separated by a hydrophilic chromatographic column, detected by a UV detector with a detection wavelength of 300 nm, and the corresponding chromatographic peak components in the preparative chromatogram are collected. The active target component is confirmed by comparing the retention time of component Fr6-3-1 with that of component Fr6-3 through high performance liquid chromatography (HPLC) analysis, and the target component Fr6-3-1 is obtained by vacuum concentration at a vacuum degree of 150 mbar and a temperature of 50°C. The working parameters of the hydrophilic high-pressure preparative column preparation of the Fr6-3 component are as follows: preparative column length 250 mm, diameter 20 mm, hydrophilic chromatographic column stationary phase 5 μm HILIC-D hydrophilic packing, mobile phase A 0.2% trifluoroacetic acid-water solution, mobile phase B acetonitrile solution, elution according to 0-60 min, 82% B, sample volume 4 mL, flow rate 19 mL / min; high performance liquid chromatography analysis comparison, using Odyssil C18 (250×4.6 mm, 5 μm) chromatographic column, detection wavelength 300 nm, using mobile phase A 0.2% trifluoroacetic acid-water solution, mobile phase B acetonitrile solution, according to 0-30 min, 5%-30% B, mobile phase flow rate 1.0 mL / min; Step 6, gel filtration chromatography: the component Fr6-3-1 is dissolved in a methanol solution to prepare a sample concentration of 100.0 mg / mL, filtered through a 0.45 μm microporous filter to obtain a filtrate C, and the filtrate C is desalted, deacidified and removed of small molecular impurities by a gel chromatographic column. The components are detected by a UV detector with a detection wavelength of 300 nm, and the main chromatographic peak components in the preparative chromatogram are collected. Each component is vacuum-concentrated at a vacuum degree of 150 mbar and a temperature of 50°C to obtain 30.25 mg of the target new compound with a purity of more than 95%, named Lycibarbarspermidines Q, with a chemical structural formula (see attached Figure 19 ) wherein the vacuum concentration conditions are a vacuum degree of 150 mbar and a temperature of 50°C; the working parameters of the gel chromatographic column are a column length of 1200 mm, a diameter of 30 mm, and a gel chromatographic column stationary phase of HW-40C, and the mobile phase B is methanol, and the chromatographic conditions are 0-1200 min, 100% B, and the flow rate is 1 mL / min.

[0020] Example 3 A separation process of a new structural α-glucosidase inhibitor Lycibarbarspermidines Q in Lycium barbarum L. specifically includes the following steps: Step 1, extraction: take 5000 g of dried red wolfberry, coarsely crush, and then extract with 100% ethanol at a material-liquid ratio of 1 g:20 mL. Extract 2 times for 4 h each time under vacuum heating reflux, filter, combine the filtrate, and vacuum reduce and concentrate the filtrate to obtain concentrated liquid A. The vacuum reduction and concentration conditions are a vacuum degree of 250 mbar and a temperature of 60°C. Step 2, online α-glucosidase binding agent component identification: add methanol to the total sample of red wolfberry at a mass of 10 times the amount of methanol to dissolve, prepare a sample concentration of 50.0 mg / mL with a volume concentration of methanol of 70%, filter through a 0.45 μm microporous filter to obtain a methanol sample solution of red wolfberry, i.e., filtrate B; take 1 mL of filtrate B and use an online HPLC-FLD chromatography system to identify the α-glucosidase inhibitor containing target component in red wolfberry (see Figure 1 The online HPLC-FLD chromatography system has a first part, a high-performance liquid chromatograph, which uses an Odyssil C18 (250x4.6 mm, 5 μm) chromatographic column and a detection wavelength of 300 nm; and a second part, a fluorescence detector and a high-performance liquid chromatograph pump, with an excitation wavelength and an emission wavelength of the fluorescence detector being 219 nm and 332 nm, respectively, and the high-performance liquid chromatograph pump pumping an α-glucosidase aqueous solution at a concentration of 15 μg / mL at a flow rate of 1 mL / min. The high-performance liquid chromatograph uses a mobile phase A of 0.2% trifluoroacetic acid-water solution and a mobile phase B of acetonitrile solution, with a flow rate of 1.0 mL / min according to 0-30 min, 5%-30% B. Step 3, hydrophilic medium pressure preparation column crude separation: wet sample the filtrate B and inject it into a medium pressure chromatographic column filled with hydrophilic filler, and use a chromatographic column to separate the sample. In the separation process, use a UV detector with a wavelength of 300 nm to monitor the effluent, and according to the detection results, collect the target chromatographic peak components in the preparation chromatogram (see Figure 2 Step 3, hydrophilic medium pressure preparation column crude separation: wet sample the filtrate B and inject it into a medium pressure chromatographic column filled with hydrophilic filler, and use a chromatographic column to separate the sample. In the separation process, use a UV detector with a wavelength of 300 nm to monitor the effluent, and according to the detection results, collect the target chromatographic peak components in the preparation chromatogram (see Figure 3The target component Fr6-3 was obtained by vacuum drying treatment after the component passed through the reverse-phase high-pressure preparation column. The working parameters of the reverse-phase high-pressure preparation column separation of the component Fr6 were as follows: the column length of the preparation column was 250 mm, the column diameter was 20 mm, the stationary phase of the reverse-phase chromatographic column was 5 μm water-resistant C18, the mobile phase A was a 0.2% trifluoroacetic acid-water solution, the mobile phase B was a methanol solution, elution was performed at 10%-35% B at 0-70 min, the injection volume was 4 mL, and the flow rate was 19 mL / min. The analysis and comparison were performed by using an Odyssil C18 (250 x 4.6 mm, 5 μm) chromatographic column, the detection wavelength was 300 nm, the mobile phase A was a 0.2% trifluoroacetic acid-water solution, the mobile phase B was an acetonitrile solution, 5%-30% B was used at 0-30 min, and the flow rate of the mobile phase was 1.0 mL / min; Step 5, hydrophilic high-pressure preparation column preparation: the component Fr6-3 was separated by a hydrophilic chromatographic column, and was detected by a UV detector with a detection wavelength of 300 nm. The target component Fr6-3 was obtained by collecting the corresponding chromatographic peak component in the preparation chromatogram (see FIG. 6). Figure 5 The active target component was confirmed, and 1.01 g of the target component Fr6-3 was obtained by vacuum drying treatment (see FIG. 6). Figure 4 The working parameters of the reverse-phase high-pressure preparation column separation of the component Fr6 were as follows: the column length of the preparation column was 250 mm, the column diameter was 20 mm, the stationary phase of the reverse-phase chromatographic column was 5 μm water-resistant C18, the mobile phase A was a 0.2% trifluoroacetic acid-water solution, the mobile phase B was a methanol solution, elution was performed at 10%-35% B at 0-70 min, the injection volume was 4 mL, and the flow rate was 19 mL / min. The analysis and comparison were performed by using an Odyssil C18 (250 x 4.6 mm, 5 μm) chromatographic column, the detection wavelength was 300 nm, the mobile phase A was a 0.2% trifluoroacetic acid-water solution, the mobile phase B was an acetonitrile solution, 5%-30% B was used at 0-30 min, and the flow rate of the mobile phase was 1.0 mL / min; Step 5, hydrophilic high-pressure preparation column preparation: the component Fr6-3 was separated by a hydrophilic chromatographic column, and was detected by a UV detector with a detection wavelength of 300 nm. The target component Fr6-3 was obtained by collecting the corresponding chromatographic peak component in the preparation chromatogram (see FIG. 6). Figure 6The active target component was confirmed by comparing the retention time of component Fr6-3-1 with that of component Fr6-3 by HPLC analysis, and the target component Fr6-3-1 was obtained by vacuum concentration under reduced pressure, wherein the vacuum concentration under reduced pressure was performed at a vacuum degree of 250 mbar and a temperature of 60°C; the working parameters of the hydrophilic high-pressure preparation column used for the Fr6-3 component were as follows: a column length of 250 mm, a column diameter of 20 mm, a hydrophilic column stationary phase of 5 μm HILIC-D hydrophilic packing material, a mobile phase A of 0.2% trifluoroacetic acid-water solution, a mobile phase B of acetonitrile solution, elution at 82% B for 0-60 min, a sample volume of 4 mL, and a flow rate of 19 mL / min; the HPLC analysis comparison was performed using an Odyssil C18 (250×4.6 mm, 5 μm) column, a detection wavelength of 300 nm, a mobile phase A of 0.2% trifluoroacetic acid-water solution, a mobile phase B of acetonitrile solution, elution at 5%-30% B for 0-30 min, and a mobile phase flow rate of 1.0 mL / min; Step 6: Gel filtration chromatography: The component Fr6-3-1 was dissolved in a methanol solution to prepare a sample at a concentration of 100.0 mg / mL, filtered through a 0.45 μm microporous filter to obtain a filtrate C, and then subjected to desalting, deacidification and removal of small molecular impurities by a gel chromatography column, detected by a UV detector at a detection wavelength of 300 nm, and the main chromatographic peak component in the preparative chromatogram was collected (see FIG. 6B). Figure 7 The components were vacuum-concentrated under reduced pressure to obtain 134.33 mg of the target new compound with a purity of greater than 95%, named Lycibarbarspermidines Q, having a chemical structural formula (see FIG. 6C for details). Figure 19 The vacuum concentration under reduced pressure was performed at a vacuum degree of 250 mbar and a temperature of 60°C; the working parameters of the gel chromatography column were as follows: a column length of 1200 mm, a column diameter of 30 mm, a gel chromatography column stationary phase of HW-40C, a mobile phase B of methanol, and a flow rate of 1 mL / min.

[0021] Example 4 Activity verification of the new structural α-glucosidase inhibitor Lycibarbarspermidines Q in Lycium ruthenicum Murr. The new structure α-glucosidase inhibitor Lycibarbarspermidines Q isolated from red medlar is dissolved in chromatographic methanol with 4 times of its mass, and a sample solution of the new structure α-glucosidase inhibitor in red medlar is prepared by filtering 1 mL of the sample solution through a 0.45 μm microporous filter at a concentration of 0.2 mg / mL, and the activity of the new structure α-glucosidase inhibitor Lycibarbarspermidines Q in red medlar is verified by using an online HPLC-FLD chromatography system; The online HPLC-FLD chromatography system comprises a first part of a high-performance liquid chromatograph, an Odyssil (250×4.6 mm, 5 μm) chromatographic column, a first high-performance liquid chromatograph, a mobile phase A of 0.2% trifluoroacetic acid-water solution, a mobile phase B of acetonitrile solution, 5-30% B at 0-30 min, a mobile phase flow rate of 1.0 mL / min, and a detection wavelength of 300 nm; and a second part of a fluorescence detector and a high-performance liquid chromatograph pump, an excitation wavelength and an emission wavelength of the fluorescence detector being 219 nm and 332 nm respectively, an α-glucosidase aqueous solution with a concentration of 15 μg / mL pumped by the high-performance liquid chromatograph pump, and a mobile phase flow rate of 1.0 mL / min. Figure 8 The activity verification chromatogram is shown in FIG. 1.

[0022] Therefore, the new structure α-glucosidase inhibitor Lycibarbarspermidines Q prepared by isolation from red medlar is applied in the preparation of a hypoglycemic drug or a health food, and the α-glucosidase inhibitor Lycibarbarspermidines Q is used as an effective component to be prepared into various pharmaceutical preparations by a conventional method together with any pharmaceutically acceptable carrier, or used as an effective component to be prepared into various health foods by a conventional method together with any food scientifically acceptable carrier.

[0023] Example 5 In-vitro inhibition experiment of the new structure α-glucosidase inhibitor Lycibarbarspermidines Q in red medlar In the microwell plate, 10 μL of the target new compound sample solution diluted with PBS buffer, 20 μL of α-glucosidase solution prepared with 0.2 U / mL and PBS buffer, 10 μL of pNPG solution prepared with 3.125 mM and distilled water, and 70 μL of PBS buffer were sequentially added to make the final reaction concentration of the sample 0.25, 0.50, 1.00, 2.00, 3.00, and 4.00 mM, respectively, and incubated at 37°C for 15 min. Then, 80 μL of NaCO3 solution prepared with 0.2 M and distilled water was added to terminate the reaction, and incubated at 37°C for 15 min. The absorbance value was measured at 405 nm. Acarbose was used as a positive control, and the blank group used an equal amount of PBS buffer instead of the sample solution, the blank control group used an equal amount of PBS buffer instead of the sample and enzyme solution, and the sample control group used an equal amount of PBS buffer instead of the enzyme solution (see Table 1). Figure 9 The inhibition rate of the sample on α-glucosidase was as follows: In the formula, ODs, ODs0, OD b , and OD b0 represent the absorbance values of the sample group, the sample control group, the blank group, and the blank control group, respectively.

[0024] Although the embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, alternatives, and variations can be made thereto without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A process for isolating Lycibarbarspermidines Q, a novel α-glucosidase inhibitor from red goji berries, characterized in that, Specifically, the steps include the following: Step 1, Extraction: Dry the red goji berries in the shade, coarsely crush them, and extract them with 100% ethanol at a material-to-liquid ratio of 1g:10~20mL. Extract under vacuum heating and reflux for 2~4 times, each time for 2~4 hours. Filter, combine the filtrates, and concentrate the filtrate under vacuum to obtain concentrated solution A. Step 2, Online Identification of α-glucosidase Inhibitor Components: Dissolve 5-10 times the mass of red goji berry in methanol (70%-90% by volume) to prepare a sample concentration of 50.0-100.0 mg / mL. Filter the solution through a 0.45 μm microporous membrane to obtain a red goji berry methanol sample solution, i.e., filtrate B. Take 1 mL of filtrate B and use an online HPLC-FLD chromatography system to identify the α-glucosidase inhibitor containing the target component in the red goji berry. The online HPLC-FLD chromatography system consists of two parts: a high-performance liquid chromatograph using an Odyssil C18 (250×4.6 mm, 5 μm) column with a detection wavelength of 300 nm; and a fluorescence detector and a high-performance liquid chromatography pump. The excitation and emission wavelengths of the fluorescence detector are 219 nm and 332 nm, respectively. The high-performance liquid chromatography pump pumps in an α-glucosidase aqueous solution. Step 3, hydrophilic medium-pressure preparative column coarse separation: Filtrate B is wet-loaded into a medium-pressure chromatographic column packed with hydrophilic packing material. The sample is separated using the chromatographic column. During the separation process, the eluent is monitored using a 300 nm ultraviolet detector. Based on the detection results, the target chromatographic peak component in the preparative chromatogram is collected. The retention time of component Fr6 is compared with that of the total red goji berry sample by high-performance liquid chromatography (HPLC) to confirm the active target component. After vacuum drying, the target component Fr6 can be obtained. Step 4, Preparation of reversed-phase high-pressure column: The component Fr6 is separated by a reversed-phase chromatographic column and detected by a UV detector with a detection wavelength of 300 nm. The corresponding chromatographic peak component Fr6-3 in the preparation chromatogram is collected and analyzed by high-performance liquid chromatography (HPLC). The retention time of component Fr6-3 is compared with that of component Fr6 to confirm the active target component. The target component Fr6-3 is obtained by vacuum drying. Step 5, preparation of hydrophilic high-pressure column: The component Fr6-3 is separated by a hydrophilic chromatographic column and detected by a UV detector with a detection wavelength of 300 nm. The corresponding chromatographic peak components in the preparation chromatogram are collected and analyzed by high performance liquid chromatography (HPLC). The retention times of component Fr6-3-1 and component Fr6-3 are compared to confirm the active target component. The target component Fr6-3-1 is obtained by vacuum drying. Step 6, Gel Filtration Chromatography: The component Fr6-3-1 was dissolved in methanol solution to prepare a sample concentration of 100.0 mg / mL. The solution was filtered through a 0.45 μm microporous membrane to obtain filtrate C. Filtrate C was then subjected to gel chromatography for desalting, acid removal, and removal of small molecule impurities. The sample was detected using a 300 nm UV detector. The main chromatographic peak components in the preparative chromatogram were collected. Each component was dried under vacuum to obtain the target new compound with a purity greater than 95%, named Lycibarbarspermidines Q, with the following chemical structural formula: ； Step 7, In vitro α-glucosidase inhibition experiment: Add 10 μL of the target compound sample solution diluted with PBS buffer, 20 μL of α-glucosidase solution prepared with 0.2 U / mL PBS buffer, 10 μL of pNPG solution prepared with 3.125 mM distilled water, and 70 μL of PBS buffer to a microplate sequentially, so that the final reaction concentrations of the samples are 0.25, 0.50, 1.00, 2.00, 3.00, and 4.00 mM, respectively. Incubate at 37℃ for 15 min, add 80 μL of NaCO3 solution prepared with 0.2 M distilled water to terminate the reaction, incubate at 37℃ for 15 min, and measure the absorbance at 405 nm. Acarbose was used as a positive control. The blank group used an equal volume of PBS buffer instead of the sample solution, the blank control group used an equal volume of PBS buffer instead of the sample and enzyme solution, and the sample control group used an equal volume of PBS buffer instead of the enzyme solution. The inhibition rate of the samples against α-glucosidase is as follows: In the formula, ODs, ODs0, and OD b OD b0 These represent the absorbance values ​​of the sample group, sample control group, blank group, and blank control group, respectively.

2. The separation process according to claim 1, characterized in that: In steps 1, 3, 4, 5, and 6, the conditions for vacuum concentration are: vacuum degree 50~250 mbar and temperature 40~60℃.

3. The separation process according to claim 1, characterized in that: In step 2, the high-performance liquid chromatograph uses a mobile phase A of 0.2% trifluoroacetic acid-water solution and a mobile phase B of acetonitrile solution, with a flow rate of 1.0 mL / min for 0-30 min, 5%-30% B; the high-performance liquid chromatograph pump pumps in α-glucosidase at a concentration of 15 μg / mL and a mobile phase flow rate of 1 mL / min.

4. The separation process according to claim 1, characterized in that: In step 3, the working parameters for the hydrophilic high-pressure preparative column separation are as follows: column length 460 mm, diameter 49 mm, stationary phase Click XION, mobile phase A is 0.2% trifluoroacetic acid, and mobile phase B is acetonitrile. The chromatographic conditions are 0–60 min, 100%–70% B, injection volume 50 g, and flow rate 80 mL / min. For high-performance liquid chromatography (HPLC) analysis and comparison, an Odyssil C18 (250 × 4.6 mm, 5 μm) column is used, with a detection wavelength of 300 nm. The mobile phase A is 0.2% trifluoroacetic acid-water solution, and mobile phase B is acetonitrile solution. The conditions are 0–30 min, 5%–30% B, and mobile phase flow rate 1.0 mL / min.

5. The separation process according to claim 1, characterized in that: In step 4, the operating parameters for the reversed-phase high-pressure preparation column used for component Fr6 are as follows: column length 250 mm, diameter 20 mm; stationary phase of the reversed-phase column is 5 μm water-resistant C18; mobile phase A is 0.2% trifluoroacetic acid-water solution; mobile phase B is methanol solution; elution is performed at 10%-35% B for 0-70 min; injection volume is 4 mL; flow rate is 19 mL / min. For high-performance liquid chromatography (HPLC) analysis and comparison, an Odyssil C18 (250×4.6 mm, 5 μm) column is used; detection wavelength is 300 nm; mobile phase A is 0.2% trifluoroacetic acid-water solution; mobile phase B is acetonitrile solution; elution is performed at 5%-30% B for 0-30 min; flow rate is 1.0 mL / min.

6. The separation process according to claim 1, characterized in that: In step 5, the working parameters for the hydrophilic high-pressure preparation column used for the Fr6-3 component are as follows: column length 250 mm, diameter 20 mm; stationary phase of the hydrophilic column is 5 μm HILIC-D hydrophilic packing material; mobile phase A is 0.2% trifluoroacetic acid-water solution; mobile phase B is acetonitrile solution; elution is performed at 0–60 min with 82% B; injection volume is 4 mL; flow rate is 19 mL / min. For high-performance liquid chromatography (HPLC) analysis and comparison, an Odyssil C18 (250 × 4.6 mm, 5 μm) column is used; detection wavelength is 300 nm; mobile phase A is 0.2% trifluoroacetic acid-water solution; mobile phase B is acetonitrile solution; elution is performed at 0–30 min with 5%–30% B; flow rate is 1.0 mL / min.

7. The separation process according to claim 1, characterized in that: In step 6, the operating parameters of the gel chromatography column are as follows: column length 1200 mm, diameter 30 mm, stationary phase HW-40C, mobile phase B methanol, chromatographic conditions 0~1200 min, 100% B, flow rate 1 mL / min.

8. The application of Lycibarbarspermidines Q, a novel α-glucosidase inhibitor from red goji berries, characterized in that: The novel α-glucosidase inhibitor Lycibarbarspermidines Q is prepared according to any one of claims 1-7 above. The novel α-glucosidase inhibitor Lycibarbarspermidines Q is used in the preparation of hypoglycemic drugs or health foods. The novel α-glucosidase inhibitor Lycibarbarspermidines Q can be used as an active ingredient to prepare various pharmaceutical preparations using conventional methods and any pharmaceutically acceptable carrier, or as an active ingredient to prepare various health foods using conventional methods and any food science-acceptable carrier.

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