Separation process of novel-structure pancreatic lipase inhibitor Lycirthepheylpropanoid H in red wolfberry and application of novel-structure pancreatic lipase inhibitor Lycirthepheylpropanoid H

Abstract

The invention discloses a separation process of a pancreatic lipase inhibitor Lycrutheophylpropanoid H with a new structure in red Chinese wolfberry fruits and application of the pancreatic lipase inhibitor Lycrutheophylpropanoid H with the new structure in the red Chinese wolfberry fruits. The specific preparation process comprises the following seven steps: extraction, on-line pancreatic lipase inhibitor component recognition, 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 pancreatic lipase 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 pancreatic lipase inhibitors, and particularly relates to a separation process of a new structure pancreatic lipase inhibitor Lyciruthephenylpropanoid H 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 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 the 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 pancreatic lipase inhibitor Lyciruthephenylpropanoid H in Lycium barbarum and application thereof.

[0005] The present application protects a separation process of a new structure pancreatic lipase inhibitor Lyciruthephenylpropanoid H 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 carried out 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 pancreatic lipase inhibitor component identification: add methanol with a mass of 5-10 times of the total sample of Lycium ruthenicum Murr. 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 Murr. methanol sample solution, i.e. filtrate B; take 1 mL of the filtrate B, and use an online HPLC-FLD chromatography system to identify the pancreatic lipase inhibitor containing the target component in Lycium ruthenicum Murr.; wherein the online HPLC-FLD chromatography system, the first part is a high performance liquid chromatograph, an Odyssil C18 (250*4.6 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 221 nm and 303 nm respectively, and the high performance liquid chromatograph pump is pumped into a pancreatic lipase 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, in the separation process, use a UV detector with a wavelength of 300 nm to monitor the effluent, 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 Fr5 with that of the total sample of Lycium ruthenicum Murr., confirm the active target component, and after vacuum and reduced pressure drying treatment, the target component Fr5 is obtained; Step 4, reverse phase high pressure preparation column preparation: the component Fr5 is separated by a reverse phase chromatographic column, detected by a UV detector with a detection wavelength of 300 nm, and the corresponding chromatographic peak component Fr5-1 in the preparation chromatogram is collected, through high performance liquid chromatography HPLC analysis, compare the retention time of the component Fr5-1 with that of the component Fr5, confirm the active target component, and vacuum and reduced pressure drying obtains the target component Fr5-1; Step 5, hydrophilic high pressure preparation column preparation: the component Fr5-1 is separated by a hydrophilic chromatographic column, detected by a UV detector with a detection wavelength of 300 nm, 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 Fr5-1-1 with that of the component Fr5-1, confirm the active target component, and vacuum and reduced pressure drying obtains the target component Fr5-1-1; Step 6, gel filtration chromatography: the component Fr5-1-1 was dissolved with methanol solution, the sample concentration was prepared to 100.0 mg / mL, the filtrate C was obtained by filtering through a 0.45 μm microporous filter, the filtrate C was desalted and removed of small molecule impurities by gel chromatography column, detected by a UV detector with a detection wavelength of 300 nm, the main chromatographic peak components in the preparative chromatogram were collected, and each component was dried by vacuum and reduced pressure to obtain the target new compound with a purity of more than 95%, named Lyciruthephenylpropanoid H, and its chemical structural formula is as follows: ; Step 7, in vitro pancreatic lipase inhibition experiment: in a microwell plate, 20 μL of the target new compound sample solution diluted with Tris-HCl buffer was added in sequence; 10 μL of pPNP solution with a concentration of 3.33 mM prepared by diluting 10 mM stock solution with ethanol; 20 μL of pancreatic lipase solution prepared with 5 mg / mL Tris-HCl buffer; and 150 μL of Tris-HCl buffer, so that the final reaction concentration of the sample was 0.25, 0.50, 1.00, 2.00, 3.00 and 4.00 mmol / L, respectively, and incubated at 37℃ for 30 min, the absorbance value was measured at 405 nm, and the half inhibitory concentration IC 50 was calculated using SPSS 19.0 software, with orlistat as a positive control, the blank group used an equal amount of Tris-HCl buffer instead of the sample solution, the blank control group used an equal amount of Tris-HCl buffer instead of the sample and enzyme solution, and the sample control group used an equal amount of Tris-HCl buffer instead of the enzyme solution, and the inhibition rate of the sample on pancreatic lipase was as follows: In the formula, ODs, ODs0, OD b , OD b0 respectively represent the absorbance values of the sample group, the sample control group, the blank group and the blank control group.

[0006] Further, in steps 1, 3, 4, 5, 6, the vacuum and reduced pressure concentration conditions are as follows: vacuum degree 50-250 mbar, temperature 40-60℃.

[0007] Further, in step 2, the mobile phase A used by the high performance liquid chromatograph is 0.2% trifluoroacetic acid-water solution, 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; the concentration of the pancreatic lipase pumped 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 separating 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 60 min, the injection volume is 50 g, and the flow rate is 80 mL / min; high-performance liquid chromatography analysis and comparison are performed using an Odyssil C18 (250x4.6 mm, 5 μm) column, the detection wavelength is 300 nm, the mobile phase A is 0.2% trifluoroacetic acid-water solution, the mobile phase B 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 preparing the component Fr5 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, the elution is performed in accordance with 15%-35% B in 0-45 min, the injection volume is 4 mL, and the flow rate is 19 mL / min; high-performance liquid chromatography analysis and comparison are performed using an Odyssil C18 (250x4.6 mm, 5 μm) column, the detection wavelength is 300 nm, the mobile phase A is 0.2% trifluoroacetic acid-water solution, the mobile phase B 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 preparing the component Fr5-1 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, the elution is performed in accordance with 80% B in 0-40 min, the injection volume is 4 mL, and the flow rate is 19 mL / min; high-performance liquid chromatography analysis and comparison are performed using an Odyssil C18 (250x4.6 mm, 5 μm) column, the detection wavelength is 300 nm, the mobile phase A is 0.2% trifluoroacetic acid-water solution, the mobile phase B 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, the chromatographic conditions are 100% B in 0-1200 min, and the flow rate is 1 mL / min.

[0012] The application also provides application of the new structure pancreatic lipase inhibitor Lyciruthephenylpropanoid H separated from the above-mentioned Lycium ruthenicum in preparation of a blood lipid-lowering drug or health food, and the new structure pancreatic lipase inhibitor Lyciruthephenylpropanoid H is used as an effective component to be prepared into various medicinal preparations by a conventional method and any pharmaceutically acceptable carrier, or used as an effective component to be prepared into various health foods by a conventional method and 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 wild or market-sold Lycium ruthenicum can be used, which is easy to prepare in batches; ethanol extraction is easy to operate.

[0015] (3) The application utilizes HPLC-FLD to identify functional components 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 field of chromatographic recognition of compound targeting / protein functional components, and thus, the applicant names this application in the laboratory as: online functional components 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 recognition chromatogram of the component of Lycium ruthenicum containing a target component of the application; Figure 2 It is a hydrophilic medium-pressure preparation column separation chromatogram of the ethanol extract of Lycium ruthenicum of the application; Figure 3The total sample of the component of the present application and Fr5 comparative chromatogram; Figure 4 The reversed-phase high-pressure preparative liquid chromatogram of the component Fr5 of the present application; Figure 5 The comparative chromatogram of the component Fr5 of the present application and Fr5-1; Figure 6 The hydrophilic high-pressure preparative liquid chromatogram of the component Fr5-1-1 of the present application; Figure 7 The gel filtration chromatogram of the component Fr5-1-1 of the present application; Figure 8 The chromatogram for verifying the purity and inhibitory activity on pancreatic lipase of the new structure pancreatic lipase inhibitor Lyciruthephenylpropanoid H of the present application; Figure 9 The half inhibitory concentration IC 50 of the new structure pancreatic lipase inhibitor Lyciruthephenylpropanoid H of the present application; Figure 10 The high-resolution mass spectrum of the new structure pancreatic lipase inhibitor Lyciruthephenylpropanoid H of the present application; Figure 11 The H NMR nuclear magnetic resonance spectrum of the new structure pancreatic lipase inhibitor Lyciruthephenylpropanoid H of the present application; 1 Figure 12 The C NMR nuclear magnetic resonance spectrum of the new structure pancreatic lipase inhibitor Lyciruthephenylpropanoid H of the present application; 13 Figure 13 The HSQC spectrum of the new structure pancreatic lipase inhibitor Lyciruthephenylpropanoid H of the present application; Figure 14 The HMBC spectrum of the new structure pancreatic lipase inhibitor Lyciruthephenylpropanoid H of the present application; Figure 15 The H-HCOSY spectrum of the new structure pancreatic lipase inhibitor Lyciruthephenylpropanoid H of the present application; Figure 16 The NOESY spectrum of the new structure pancreatic lipase inhibitor Lyciruthephenylpropanoid H of the present application; Figure 17 ​​The TOCSY map of the new structure of pancreatic lipase inhibitor Lyciruthephenylpropanoid H of the present application; Figure 18 The DEPT map of the new structure of pancreatic lipase inhibitor Lyciruthephenylpropanoid H of the present application; Figure 19 The planar chemical structure formula map of the new structure of pancreatic lipase inhibitor Lyciruthephenylpropanoid H 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 a part 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 of pancreatic lipase inhibitor Lyciruthephenylpropanoid H in red wolfberry, specifically comprising the following steps: Step 1, extraction: 500 g of dried red wolfberry is coarsely crushed and extracted with 100% ethanol according to the solid-liquid ratio of 1 g: 10 mL, vacuum heating and reflux extraction for 4 times, 2 h each time, filtration, and the filtrate is combined and concentrated under vacuum and reduced pressure to obtain concentrated solution A, wherein the vacuum and reduced pressure concentration conditions are vacuum degree of 50 mbar and temperature of 40℃; Step 2, online pancrelipase binding agent component identification: add methanol with 5 times of the mass of the total sample of Lycium ruthenicum Murr to dissolve, the volume concentration of methanol is 90%, prepare the sample concentration to be 100.0 mg / mL, filter through a 0.45 μm microporous filter membrane to obtain a Lycium ruthenicum Murr methanol sample solution, i.e. filtrate B; take 1 mL of the filtrate B, and use an online HPLC-FLD chromatography system to identify the pancrelipase inhibitor containing the target component in Lycium ruthenicum Murr; 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 221 nm and 303 nm respectively, the pancrelipase aqueous solution concentration pumped by the high performance liquid chromatograph pump is 15 μg / mL, and the flow rate of the mobile phase is 1 mL / min; wherein 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, according to 0-30 min, 5%-30% B, the flow rate of the mobile phase is 1.0 mL / min; 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, separate the sample by using the chromatographic column, monitor the effluent in the separation process by using a ultraviolet detector with a wavelength of 300 nm, collect the target chromatographic peak components in the preparation chromatogram according to the detection results, compare the components Fr5 with the total sample of Lycium ruthenicum Murr by high performance liquid chromatography HPLC analysis, confirm the active target component, and the target component Fr5 can be obtained after vacuum and reduced pressure drying treatment, i.e. 1.54 g; wherein the vacuum and reduced pressure concentration conditions are a vacuum degree of 50 mbar and a temperature of 40℃; the working parameters of the hydrophilic medium pressure preparation column separation are a chromatographic column length of 460 mm, a diameter of 49 mm, a stationary phase of ClickXION, a mobile phase A of 0.2% trifluoroacetic acid, a mobile phase B of acetonitrile, chromatographic conditions of 0-60 min, 100%-70% B, 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, according to 0-30 min, 5%-30% B, and a flow rate of the mobile phase of 1.0 mL / min; Step 4, reverse phase high pressure preparative column preparation: component Fr5 was separated by a reverse phase chromatographic column, detected by a UV detector with a detection wavelength of 300 nm, and component Fr5-1 corresponding to the chromatographic peak in the preparative chromatogram was collected. The active target component was confirmed by comparing the retention time of component Fr5-1 with that of component Fr5 by high performance liquid chromatography (HPLC) analysis. The target component Fr5-1 was obtained by vacuum and reduced pressure drying, wherein the vacuum and reduced pressure concentration conditions were 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 Fr5 were a preparative column length of 250 mm and 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 according to 0-45 min, 15%-35% B, a sample injection 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) 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, elution according to 0-30 min, 5%-30% B, and a mobile phase flow rate of 1.0 mL / min; Step 5, hydrophilic high pressure preparative column preparation: component Fr5-1 was separated by a hydrophilic chromatographic column, detected by a UV detector with a detection wavelength of 300 nm, and the target component was collected. The active target component was confirmed by comparing the retention time of component Fr5-1-1 with that of component Fr5-1 by high performance liquid chromatography (HPLC) analysis. The target component Fr5-1-1 was obtained by vacuum and reduced pressure drying, wherein the vacuum and reduced pressure concentration conditions were a vacuum degree of 50 mbar and a temperature of 40°C; the working parameters of the hydrophilic high pressure preparative column preparation of Fr5-1 component were a preparative column length of 250 mm and a diameter of 20 mm, a hydrophilic chromatographic column stationary phase of 5 μm HILIC-D hydrophilic filler, a mobile phase A of 0.2% trifluoroacetic acid-water solution, a mobile phase B of acetonitrile solution, elution according to 0-40 min, 80% B, a sample injection 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) 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, elution according to 0-30 min, 5%-30% B, and a mobile phase flow rate of 1.0 mL / min; Step 6, gel filtration chromatography: the component Fr5-1-1 was dissolved with methanol solution, the sample concentration was prepared to be 100.0 mg / mL, the filtrate C was obtained by filtering through a 0.45 μm microporous filter, the filtrate C was desalted and removed of small molecule impurities by gel chromatography column, detected by a UV detector with a detection wavelength of 300 nm, the main chromatographic peak components in the preparative chromatogram were collected, and each component was dried by vacuum and reduced pressure to obtain 12.34 mg of the target new compound with a purity of more than 95%, named Lyciruthephenylpropanoid H, whose chemical structural formula (see attached Figure 19 );wherein, the vacuum and reduced pressure concentration conditions were 50 mbar of vacuum degree and 40℃ of temperature; the working parameters of the gel chromatography column were 1200 mm of column length and 30 mm of diameter, the gel chromatography column stationary phase was HW-40C, the mobile phase B was methanol, and the chromatographic conditions were 100% B at 0-1200 min, and the flow rate was 1 mL / min.

[0019] Example 2 A separation process of a new structure pancreatic lipase inhibitor Lyciruthephenylpropanoid H in red wolfberry, specifically comprising the following steps: Step 1, extraction: 1000 g of red wolfberry was dried and coarsely crushed, then extracted with 100% ethanol according to a solid-liquid ratio of 1 g:15 mL, vacuum and heated to reflux for 3 times, each for 3 h, filtered, the filtrates were combined, and the filtrate was vacuum and reduced pressure concentrated to obtain concentrated liquid A, wherein the vacuum and reduced pressure concentration conditions were 150 mbar of vacuum degree and 50℃ of temperature; Step 2, online pancreatic lipase binder component identification: 8 times the mass of methanol was added to the total sample of red wolfberry for dissolution, the volume concentration of methanol was 80%, the sample concentration was prepared to be 80.0 mg / mL, and the red wolfberry methanol sample solution, i.e. the filtrate B, was obtained by filtering through a 0.45 μm microporous filter; 1 mL of the filtrate B was used to identify the pancreatic lipase inhibitor containing the target component in red wolfberry by 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) chromatography column was used, and the detection wavelength was 300 nm; the second part was a fluorescence detector and a high performance liquid chromatography pump, the excitation wavelength and the emission wavelength of the fluorescence detector were 221 nm and 303 nm respectively, the pancreatic lipase aqueous solution concentration pumped into the high performance liquid chromatography pump was 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 5%-30% B at 0-30 min, and 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 Fr5 with that of the total sample of red medlar. After vacuum and reduced pressure drying treatment, 3.44 g of target component Fr5 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 Fr5 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 Fr5-1 in the preparative chromatogram is collected. The active target component is confirmed by comparing the retention time of component Fr5-1 with that of component Fr5, and 100.32 mg of target component Fr5-1 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 Fr5 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 15%-35% B in 0-45 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 Fr5-1 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 Fr5-1-1 with that of component Fr5-1 through high performance liquid chromatography (HPLC) analysis. The target component Fr5-1-1 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 hydrophilic high-pressure preparative column preparation of the Fr5-1 component are as follows: a preparative column length of 250 mm and a diameter of 20 mm, a hydrophilic chromatographic column stationary phase of 5 μm HILIC-D hydrophilic packing, a mobile phase A of 0.2% trifluoroacetic acid-water solution, a mobile phase B of acetonitrile solution, elution of 80% B from 0 to 40 min, a sample injection volume of 4 mL, and a flow rate of 19 mL / min. The high performance liquid chromatography (HPLC) analysis comparison is performed using an Odyssil C18 (250×4.6 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, 5%-30% B from 0 to 30 min, and a mobile phase flow rate of 1.0 mL / min. Step 6, gel filtration chromatography: the component Fr5-1-1 is dissolved in a methanol solution to prepare a sample with a concentration of 100.0 mg / mL, and then filtered through a 0.45 μm microporous filter to obtain a filtrate C. The filtrate C is desalted, deacidified, and small molecular impurities are removed 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 dried by vacuum and reduced pressure to obtain 28.66 mg of a target new compound with a purity of more than 95%, which is named Lyciruthephenylpropanoid H, and has a chemical structural formula (see Figure 1 for details). Figure 19 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 gel chromatographic column are as follows: a chromatographic column length of 1200 mm and a diameter of 30 mm, a gel chromatographic column stationary phase of HW-40C, a mobile phase B of methanol, and a chromatographic condition of 100% B from 0 to 1200 min at a flow rate of 1 mL / min.

[0020] Example 3 A separation process of a new structural pancreatic lipase inhibitor Lyciruthephenylpropanoid H in Lycium ruthenicum is disclosed, which specifically comprises 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 under vacuum heating reflux, 4 h each time, 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 pancreatic lipase binder component identification: add 10 times the mass of methanol to the total sample of red wolfberry to dissolve, prepare a sample concentration of 50.0 mg / mL with a methanol volume concentration 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 pancreatic lipase inhibitor containing target components (see Figure 1 ); wherein the online HPLC-FLD chromatography system has a first part, a high-performance liquid chromatograph, which uses an Odyssil C18 (250×4.6 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, which has an excitation wavelength and an emission wavelength of 221 nm and 303 nm, respectively, for the fluorescence detector, and a concentration of 15 μg / mL of the pancreatic lipase aqueous solution pumped by the high-performance liquid chromatograph pump at a flow rate of 1 mL / min; wherein 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, and the flow rate of the mobile phase is 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 the 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 ), and compare the components Fr5 with the total sample of red wolfberry by high-performance liquid chromatography HPLC analysis to confirm the active target components (see Figure 3As shown in the figure, after vacuum drying, 16.32 g of the target component Fr5 was obtained. The vacuum concentration conditions were 250 mbar and 60℃. The working parameters for hydrophilic high-pressure preparative column separation were: column length 460 mm, diameter 49 mm, stationary phase Click XION, mobile phase A 0.2% trifluoroacetic acid, mobile phase B acetonitrile, chromatographic conditions 0-60 min, 100%-70% B, injection volume 50 g, and flow rate 80 mL / min. High-performance liquid chromatography (HPLC) analysis was performed using an Odyssil C18 (250×4.6 mm, 5 μm) column, detection wavelength 300 nm, mobile phase A 0.2% trifluoroacetic acid-water solution, mobile phase B acetonitrile solution, 0-30 min, 5%-30% B, and mobile phase flow rate 1.0 mL / min. Step 4, Preparation using a reversed-phase high-pressure column: Component Fr5 is separated using a reversed-phase column and detected by a 300 nm UV detector. The corresponding peak component Fr5-1 in the preparative chromatogram is collected and analyzed by high-performance liquid chromatography (HPLC). The retention times of component Fr5-1 and component Fr5 are compared (see attached). Figure 5 As shown), the active target component was confirmed, and 602.33 mg of the target component Fr5-1 was obtained by vacuum drying (see attached). Figure 4 As shown in the figure, the vacuum concentration conditions were a vacuum degree of 250 mbar and a temperature of 60℃. The working parameters of the reversed-phase high-pressure preparation column used for component Fr5 were as follows: column length 250 mm, diameter 20 mm, stationary phase of 5 μm water-resistant C18, mobile phase A of 0.2% trifluoroacetic acid-water solution, mobile phase B of methanol solution, elution at 15%-35% B for 0~45 min, injection volume of 4 mL, and flow rate of 19 mL / min. The high-performance liquid chromatography (HPLC) analysis and comparison were performed using an Odyssil C18 (250×4.6 mm, 5 μm) column, detection wavelength of 300 nm, mobile phase A of 0.2% trifluoroacetic acid-water solution, mobile phase B of acetonitrile solution, elution at 5%-30% B for 0~30 min, and flow rate of 1.0 mL / min. Step 5, Preparation using a hydrophilic high-pressure column: Component Fr5-1 is separated using a hydrophilic chromatographic column and detected by a 300nm UV detector. The corresponding chromatographic peak components in the preparative chromatogram are collected (see attached image). Figure 6The active target component was confirmed by comparing the retention time of component Fr5-1-1 with that of component Fr5-1 by HPLC analysis, and the target component Fr5-1-1 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 Fr5-1 component were as follows: column length of the preparation column, 250 mm; column diameter, 20 mm; hydrophilic column stationary phase, 5 μm HILIC-D hydrophilic packing material; mobile phase A, 0.2% trifluoroacetic acid aqueous solution; mobile phase B, acetonitrile solution; elution at 80% B from 0 to 40 min; sample volume, 4 mL; and flow rate, 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, mobile phase A of 0.2% trifluoroacetic acid aqueous solution, and mobile phase B of acetonitrile solution, at 5%-30% B from 0 to 30 min, and a flow rate of 1.0 mL / min; Step 6, gel filtration chromatography: the component Fr5-1-1 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 the filtrate C was 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. 6). Figure 7 The components were vacuum-concentrated under reduced pressure to obtain 150.86 mg of the target new compound with a purity of greater than 95%, which was named Lyciruthephenylpropanoid H, and the chemical structural formula thereof is shown in FIG. 7 (see FIG. 7 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; and the working parameters of the gel chromatography column were as follows: column length, 1200 mm; column diameter, 30 mm; gel chromatography column stationary phase, HW-40C; mobile phase B, methanol; and chromatographic conditions, 100% B from 0 to 1200 min, at a flow rate of 1 mL / min.

[0021] Example 4 Activity verification of the new structural pancreatic lipase inhibitor Lyciruthephenylpropanoid H in Lycium ruthenicum The new structure of pancreatic lipase inhibitor Lyciruthephenylpropanoid H is isolated from red wolfberry, and 4 times the mass of chromatographic methanol is added for dissolution. The sample concentration is 0.2 mg / mL, and the sample solution of the new structure of pancreatic lipase inhibitor is obtained by filtering through a 0.45 μm microporous filter. 1 mL of the sample is taken, and the activity of the new structure of pancreatic lipase inhibitor Lyciruthephenylpropanoid H in red wolfberry is verified by using an online HPLC-FLD chromatography system. The online HPLC-FLD chromatography system includes a high-performance liquid chromatograph, a fluorescence detector, and a high-performance liquid chromatograph pump. The high-performance liquid chromatograph uses an Odyssil (250×4.6 mm, 5 μm) chromatographic column. The mobile phase A of the first high-performance liquid chromatograph is a 0.2% trifluoroacetic acid-water solution, and the mobile phase B is an acetonitrile solution. The flow rate of the mobile phase is 1.0 mL / min, and the detection wavelength is 300 nm. The excitation wavelength and the emission wavelength of the fluorescence detector are 221 nm and 303 nm, respectively. The concentration of the pancreatic lipase aqueous solution pumped into the high-performance liquid chromatograph pump is 15 μg / mL, and the flow rate of the mobile phase is 1.0 mL / min. The activity verification chromatogram is shown in FIG. 1. Figure 8

[0022] Therefore, the new structure of pancreatic lipase inhibitor Lyciruthephenylpropanoid H prepared by the above-mentioned separation of red wolfberry is applied in the preparation of a blood lipid-lowering drug or a health food. The new structure of pancreatic lipase inhibitor Lyciruthephenylpropanoid H is used as an effective component to prepare various pharmaceutical preparations by a conventional method with any pharmaceutically acceptable carrier, or is used as an effective component to prepare various health foods by a conventional method with any food scientifically acceptable carrier.

[0023] Example 5 In vitro inhibition experiment of the new structure of pancreatic lipase inhibitor Lyciruthephenylpropanoid H in red wolfberry: ​In the microwell plate, 20 μL of the target new compound sample solution diluted with Tris-HCl buffer, 10 μL of pPNP solution diluted with ethanol to 3.33 mM, 20 μL of lipase solution diluted with 5 mg / mL Tris-HCl buffer, and 150 μL of Tris-HCl 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 mmol / L, respectively, and incubated at 37°C for 30 min. The absorbance value was measured at 405 nm, and the half inhibitory concentration IC50 of different lipase inhibitors was calculated using SPSS 19.0 software. 50 Orlistat was used as a positive control, the blank group used an equal amount of Tris-HCl buffer instead of the sample solution, the blank control group used an equal amount of Tris-HCl buffer instead of the sample and enzyme solution, and the sample control group used an equal amount of Tris-HCl buffer instead of the enzyme solution (see Table 1). Figure 9 The inhibition rate of the sample on lipase was as follows: In the formula, ODs, ODs0, OD b , OD b0 respectively represent the absorbance values of the sample group, the sample control group, the blank group and the blank control group.

[0024] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments 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 Lyciruthephenylpropanoid H, a novel structural pancreatic lipase inhibitor from red wolfberry, 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 Pancreatic Lipase 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 target component, the pancreatic lipase inhibitor, 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 221 nm and 303 nm, respectively. The high-performance liquid chromatography pump pumps in an aqueous solution of pancreatic lipase. 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, a 300 nm ultraviolet detector is used to monitor the eluent. Based on the detection results, the target chromatographic peak component in the preparative chromatogram is collected. The retention time of component Fr5 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 Fr5 can be obtained. Step 4, reversed-phase high-pressure preparation column preparation: The component Fr5 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 Fr5-1 in the preparation chromatogram is collected and analyzed by high-performance liquid chromatography (HPLC). The retention time of component Fr5-1 is compared with that of component Fr5 to confirm the active target component. The target component Fr5-1 is obtained by vacuum drying. Step 5, preparation of hydrophilic high-pressure column: The component Fr5-1 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 time of component Fr5-1-1 is compared with that of component Fr5-1 to confirm the active target component. The target component Fr5-1-1 is obtained by vacuum drying. Step 6, Gel filtration chromatography: The component Fr5-1-1 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 subjected to gel chromatography column desalting, acid removal, and removal of small molecule impurities. The sample was detected by a 300 nm UV detector. The main chromatographic peak components in the prepared chromatogram were collected. Each component was dried under vacuum to obtain the target new compound with a purity greater than 95%, named Lyciruthephenylpropanoid H, with the following chemical structural formula: ； Step 7, In vitro pancreatic lipase inhibition experiment: Add 20 μL of the target compound sample solution diluted with Tris-HCl buffer to a microplate sequentially; 10 μL of a 10 mM stock solution prepared with acetonitrile, diluted to 3.33 mM with ethanol before use; 20 μL of pancreatic lipase solution prepared with 5 mg / mL Tris-HCl buffer; and 150 μL of Tris-HCl buffer to achieve final reaction concentrations of 0.25, 0.50, 1.00, 2.00, 3.00, and 4.00 mmol / L, respectively. Incubate at 37°C for 30 min, measure the absorbance at 405 nm, and calculate the half-maximal inhibitory concentration (IC50) of different pancreatic lipase inhibitors using SPSS 19.0 software. 50 Orlistat was used as a positive control. The blank control group used an equal volume of Tris-HCl buffer instead of the sample solution, the blank control group used an equal volume of Tris-HCl buffer instead of the sample and enzyme solution, and the sample control group used an equal volume of Tris-HCl buffer instead of the enzyme solution. The inhibition rates of the samples against pancreatic lipase are 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 injects pancreatic lipase at a concentration of 15 μg / mL, with 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 Fr5 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 15%-35% B for 0-45 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 Fr5-1 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 0–40 min with 80% 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 a novel structural pancreatic lipase inhibitor, Lyciruthephenylpropanoid H, from red wolfberry, characterized in that: The novel pancreatic lipase inhibitor Lyciruthephenylpropanoid H is prepared according to any one of claims 1-7. The novel pancreatic lipase inhibitor Lyciruthephenylpropanoid H is used in the preparation of lipid-lowering drugs or health foods. The novel pancreatic lipase inhibitor Lyciruthephenylpropanoid H is 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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