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

Abstract

The invention discloses a separation process of a tyrosinase inhibitor Lycirthepheylpropanoid H with a new structure in lycium barbarum and application of the tyrosinase inhibitor Lycirthepheylpropanoid H with the new structure in lycium barbarum. The specific preparation process comprises the following seven steps: extraction, on-line tyrosinase 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 tyrosinase 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 invention relates to the technical field of separation of tyrosinase inhibitors, and in particular to a separation process of Lyciruthephenylpropanoid H, a tyrosinase inhibitor with a new structure in red wolfberry, and application thereof. Background Art

[0002] Lycium barbarum L., a plant of the genus Lycium in the Solanaceae family, is primarily distributed in northwest China and has a long history of medicinal and edible use. Its fruit is rich in a variety of bioactive components, including polysaccharides, carotenoids, flavonoids, vitamins, and minerals. In traditional Chinese medicine, red wolfberry is widely used for liver and kidney nourishment, eye health, anti-aging, and immune enhancement. Modern research indicates that red wolfberry, rich in polysaccharides, flavonoids, carotenoids, phenolic acids, and alkaloids, exhibits pharmacological activities such as anti-inflammatory, antioxidant, hypoglycemic, immunomodulatory, and neuroprotective properties, suggesting significant potential for development. Literature reports indicate that phenylpropanoids and flavonoids (caffeic acid, quercetin-3-O-rutinoside, and kaempferol-3-O-rutinoside) in red wolfberry exhibit antioxidant properties. The seven different types of polyphenolic compounds in wolfberry, including phenylpropanoids, coumarins, lignans, flavonoids, isoflavones, chlorogenic acid, p-hydroxybenzaldehyde and p-hydroxybenzoic acid compounds, have antioxidant and free radical scavenging capabilities.

[0003] As a natural product with rich biologically active ingredients, red wolfberry needs to explore more active ingredients in order to further accelerate the quality evaluation, production and sales of red wolfberry and the research and development of related new drugs. Summary of the Invention

[0004] Based on the above technical problems, the purpose of the present invention is to provide a separation process and application of Lyciruthephenylpropanoid H, a new structure tyrosinase inhibitor in red wolfberry.

[0005] The present invention provides a process for separating Lyciruthephenylpropanoid H, a novel tyrosinase inhibitor from red wolfberry, which specifically comprises the following steps: Step 1, extraction: Dry the red wolfberry in the shade, coarsely chop it, and extract it with 100% ethanol at a material-liquid ratio of 1 g: 10-20 mL. Heat under vacuum reflux for 2-4 times, each time for 2-4 hours. Filter, combine the filtrates, and concentrate the filtrates under vacuum to obtain concentrate A. Step 2, online tyrosinase inhibitor component identification: 5 to 10 times the mass of methanol was added to the total red wolfberry sample to dissolve it, the volume concentration of the methanol being 70% to 90%, and the sample concentration being prepared to be 50.0 to 100.0 mg / mL. The sample was filtered through a 0.45 μm microporous filter membrane to obtain a red wolfberry methanol sample solution, i.e., filtrate B. 1 mL of filtrate B was taken and used to identify the tyrosinase inhibitor containing the target component in the red wolfberry using an online HPLC-FLD chromatography system. The online HPLC-FLD chromatography system comprised a high performance liquid chromatograph, a first part using an Odyssil C18 (250×4.6 mm, 5 μm) column, and a detection wavelength of 300 nm. The second part comprised a fluorescence detector and a high performance liquid chromatography pump, the excitation wavelength and emission wavelength of the fluorescence detector being 221 nm and 331 nm, respectively, and the high performance liquid chromatography pump pumped the tyrosinase aqueous solution. Step 3, hydrophilic medium-pressure preparative column crude fractionation: Filtrate B is wet-loaded and injected into a medium-pressure chromatographic column filled with hydrophilic filler. The sample is separated using the chromatographic column. During the separation process, the effluent is monitored using a UV detector with a wavelength of 300 nm. Based on the detection results, the target chromatographic peak components in the preparative chromatogram are collected and analyzed by high-performance liquid chromatography (HPLC). The retention time of component Fr5 is compared with that of the total red wolfberry sample to confirm the active target component. After the component is dried under vacuum, the target component Fr5 can be obtained. Step 4, reverse-phase high-pressure preparative column preparation: The 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 component Fr5-1 is analyzed by high-performance liquid chromatography (HPLC), and the retention time of the component Fr5-1 is compared with that of the component Fr5 to confirm the active target component. The target component Fr5-1 is obtained by vacuum drying. Step 5, preparation of a hydrophilic high-pressure preparative column: The component Fr5-1 is separated by a hydrophilic chromatographic column and detected by an ultraviolet detector with a detection wavelength of 300 nm. The corresponding chromatographic peak components in the preparative 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, and the sample was filtered through a 0.45 μm microporous filter membrane to obtain filtrate C. Filtrate C was desalted, deacidified, and small molecular impurities were removed by gel chromatography column. The filtrate C was detected by an ultraviolet detector with a detection wavelength of 300 nm. The main chromatographic peak components in the preparative chromatogram were collected and each component was dried under vacuum to obtain a target new compound with a purity greater than 95%, named Lyciruthephenylpropanoid H, whose chemical structure is as follows: ; Step 7, in vitro tyrosinase inhibition assay: 40 μL of a sample solution of the target new compound diluted with PBS buffer at appropriate concentrations was added to the microplate; 20 μL of a 0.2 mg / mL L-tyrosine solution prepared with PBS buffer and 30 μL of Tris-HCl buffer were added in sequence, so that the final reaction concentrations of the samples were 0.25, 0.50, 1.00, 2.00, 3.00 and 4.00 mmol / L, respectively. The samples were incubated at 37°C for 10 min, and 40 μL of a tyrosinase solution prepared with 98.5 μg / mL PBS buffer was added. The samples were incubated at 37°C for 10 min, and the absorbance was measured at 475 nm. The half-maximal inhibitory concentration (IC) of different tyrosinase inhibitors was calculated using SPSS 19.0 software. 50 Kojic acid was used as the positive control. The blank group used an equal amount of PBS buffer to replace the sample solution. The blank control group used an equal amount of PBS buffer to replace the sample and enzyme solution. The sample control group used an equal amount of PBS buffer to replace the enzyme solution. The inhibition rate of the sample on tyrosinase is as follows: Where, ODs, ODs0, OD b ,OD b0 Represent the absorbance values ​​of the sample group, sample control group, blank group and blank control group, respectively.

[0006] Furthermore, in step 1, step 3, step 4, step 5, and step 6, the conditions for vacuum decompression and concentration are: vacuum degree 50-250 mbar, temperature 40-60°C.

[0007] Furthermore, in step 2, the mobile phase A used in 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, and the mobile phase flow rate is 1.0 mL / min; the tyrosinase concentration pumped into the high performance liquid chromatography pump is 15 μg / mL, and the mobile phase flow rate is 1 mL / min.

[0008] Furthermore, in step 3, the working parameters of the hydrophilic preparative column separation are as follows: a chromatographic column length of 460 mm and a diameter of 49 mm, a stationary phase of Click XION, a mobile phase A of 0.2% trifluoroacetic acid, B of acetonitrile, and chromatographic conditions of 0-60 min, 100%-70% B, an injection volume of 50 g, and a flow rate of 80 mL / min; for high performance liquid chromatography analysis and comparison, an Odyssil C18 (250 × 4.6 mm, 5 μm) column was used, the detection wavelength was 300 nm, the mobile phase A used was 0.2% trifluoroacetic acid-water solution, the mobile phase B was acetonitrile solution, according to 0-30 min, 5%-30% B, and the mobile phase flow rate was 1.0 mL / min.

[0009] Furthermore, in step 4, the working parameters for the preparation of the reversed-phase high-pressure preparative column used for the component Fr5 are as follows: a preparative column length of 250 mm and a diameter of 20 mm, a reversed-phase chromatographic column stationary phase of 5 μm pure water-resistant C18, mobile phase A of 0.2% trifluoroacetic acid-water solution, mobile phase B of methanol solution, eluted according to 0-45 min, 15%-35% B, an injection volume of 4 mL, and a flow rate of 19 mL / min; for high performance liquid chromatography analysis and comparison, an Odyssil C18 (250×4.6 mm, 5 μm) column was used, the detection wavelength was 300 nm, the mobile phase A used was 0.2% trifluoroacetic acid-water solution, the mobile phase B was acetonitrile solution, eluted according to 0-30 min, 5%-30% B, and the mobile phase flow rate was 1.0 mL / min.

[0010] Furthermore, in step 5, the working parameters for the preparation of the hydrophilic high-pressure preparative column used for the Fr5-1 component are as follows: the preparative column has a length of 250 mm and a diameter of 20 mm, the hydrophilic chromatographic column stationary phase is a 5 μm HILIC-D hydrophilic filler, the mobile phase A is 0.2% trifluoroacetic acid-water solution, the mobile phase B is an acetonitrile solution, according to 0~40 min, 80% B elution, the injection volume is 4 mL, and the flow rate is 19 mL / min; for high performance liquid chromatography analysis and comparison, an Odyssil C18 (250×4.6 mm, 5 μm) column is used, the detection wavelength is 300 nm, the mobile phase A is 0.2% trifluoroacetic acid-water solution, the mobile phase B is acetonitrile solution, according to 0~30 min, 5%-30% B, and the mobile phase flow rate is 1.0 mL / min.

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

[0012] The present invention also provides the use of the novel tyrosinase inhibitor Lyciruthephenylpropanoid H isolated from the red wolfberry in the preparation of drugs or health foods for reducing melanin production. The novel tyrosinase inhibitor Lyciruthephenylpropanoid H is used as an active ingredient and is prepared into various pharmaceutical preparations according to conventional methods with any pharmaceutically acceptable carrier, or is used as an active ingredient and is prepared into various health foods according to conventional methods with any carrier acceptable in food science.

[0013] Compared with the existing technology, the present invention has the following beneficial effects: (1) The present invention has low cost and high product purity The extraction solvent, hydrophilic preparative column, reversed-phase preparative column, gel chromatography, and solvents and separation materials used in the reversed-phase chromatographic column in the preparation process of the present invention can all be recycled; the chromatographic separation materials used can all be reused, and the recycled solvent and reused separation materials ensure that the average cost of the separation process is relatively low. High-pressure chromatographic separation can ensure that the purity of the product is greater than 95%.

[0014] (2) The preparation method of the present invention can realize the needs of large-scale production The raw material requirements are not high and the cost is low. Generally, wild or commercially available red wolfberries can be used, which are easy to prepare in batches; ethanol extraction is easy to operate.

[0015] (3) The present invention uses HPLC-FLD to identify functional components High-performance liquid chromatography-fluorescence detection (HPLC-FLD) is an analytical method that combines high-performance liquid chromatography separation technology, continuous pumping of enzyme solution, and fluorescence detection. The present invention applies this technology to the field of chromatographic identification of targeted compound / protein functional components for the first time. Therefore, the applicant has named this application of this technology in the laboratory: Online functional components chromatographic recognition system for target enzymes / proteins, abbreviated as HPLC-FLD or UV-FLD. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is an online HPLC-FLD identification chromatogram of components containing target components in red wolfberry of the present invention; Figure 2 This is a chromatogram of the hydrophilic preparative column separation of the red wolfberry ethanol extract of the present invention; Figure 3 This is a comparative chromatogram of the total sample of the components of the present invention and Fr5; Figure 4 This is the reverse phase high pressure preparative liquid chromatogram of component Fr5 of the present invention; Figure 5 This is a comparative chromatogram of components Fr5 and Fr5-1 of the present invention; Figure 6 This is the hydrophilic high pressure preparative liquid chromatogram of component Fr5-1-1 of the present invention; Figure 7 This is the gel filtration chromatography chromatogram of component Fr5-1-1 of the present invention; Figure 8 The figure is a chromatogram showing the purity and inhibitory activity of the novel tyrosinase inhibitor Lyciruthephenylpropanoid H on tyrosinase of the present invention; Figure 9 is the half maximal inhibitory concentration IC of the novel tyrosinase inhibitor Lyciruthephenylpropanoid H of the present invention 50 ; Figure 10 This is a high-resolution mass spectrum of the novel tyrosinase inhibitor Lyciruthephenylpropanoid H of the present invention; Figure 11 The novel structure tyrosinase inhibitor Lyciruthephenylpropanoid H of the present invention 1 H NMR spectrum; Figure 12 The novel structure of the tyrosinase inhibitor Lyciruthephenylpropanoid H of the present invention 13 C NMR spectra; Figure 13 HSQC spectrum of the novel tyrosinase inhibitor Lyciruthephenylpropanoid H of the present invention; Figure 14 The HMBC spectrum of the novel tyrosinase inhibitor Lyciruthephenylpropanoid H of the present invention; Figure 15 The H-HCOSY spectrum of the novel tyrosinase inhibitor Lyciruthephenylpropanoid H of the present invention; Figure 16 The NOESY spectrum of the novel tyrosinase inhibitor Lyciruthephenylpropanoid H of the present invention; Figure 17 The TOCSY spectrum of the novel tyrosinase inhibitor Lyciruthephenylpropanoid H of the present invention; Figure 18 The DEPT spectrum of the novel tyrosinase inhibitor Lyciruthephenylpropanoid H of the present invention; Figure 19 The planar chemical structure diagram of the novel tyrosinase inhibitor Lyciruthephenylpropanoid H of the present invention. DETAILED DESCRIPTION

[0017] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0018] Example 1 A process for separating Lyciruthephenylpropanoid H, a novel tyrosinase inhibitor from red wolfberry, comprises the following steps: Step 1, extraction: 500 g of red wolfberry was dried in the shade, coarsely chopped, and extracted with 100% ethanol at a material-liquid ratio of 1 g:10 mL. The extraction was performed under vacuum heating and reflux for 4 times, each time for 2 h. The filtrates were filtered, combined, and concentrated under vacuum to obtain a concentrate A. The vacuum concentration conditions were 50 mbar and 40°C. Step 2, online identification of tyrosinase binder components: 5 times the mass of methanol was added to the total sample of red wolfberry to dissolve it, and the volume concentration of methanol was 90%. The sample concentration was prepared to 100.0 mg / mL, and filtered through a 0.45 μm microporous filter membrane to obtain a red wolfberry methanol sample solution, i.e., filtrate B; 1 mL of filtrate B was taken and used to identify the tyrosinase inhibitor containing the target component in red wolfberry using an online HPLC-FLD chromatography system; wherein, the online HPLC-FLD chromatography system, the first part of which is a high performance liquid chromatograph, using an Odyssil C18 (250×4.6 mm, 5 μm) column, and a detection wavelength of 300 nm; the second part is a fluorescence detector and a high performance liquid chromatography pump, the excitation wavelength and emission wavelength of the fluorescence detector are 221 nm and 331 nm, respectively, the concentration of the tyrosinase aqueous solution pumped into the high performance liquid chromatography pump is 15 μg / mL, and the flow rate of the mobile phase is 1 mL / min; the mobile phase A used in the high performance liquid chromatography was 0.2% trifluoroacetic acid-water solution, and the mobile phase B was acetonitrile solution. The flow rate of the mobile phase was 1.0 mL / min according to the flow rate of 5%-30% B from 0 to 30 min. Step 3, hydrophilic preparative column crude fractionation: filtrate B was wet loaded and injected into a medium-pressure chromatographic column filled with hydrophilic filler, and the sample was separated by the chromatographic column. During the separation process, the effluent was monitored by a UV detector with a wavelength of 300 nm. According to the detection results, the target chromatographic peak components in the preparative chromatogram were collected and analyzed by high-performance liquid chromatography (HPLC). The retention time of component Fr5 was compared with that of the total red wolfberry sample to confirm the active target component. After vacuum decompression and drying, 1.54 g of the target component Fr5 was obtained. The vacuum decompression and concentration conditions were a vacuum degree of 50 mbar and a temperature of 40°C. The operating parameters of the hydrophilic preparative column separation were as follows: the column length was 460 mm and the diameter was 49 mm, the stationary phase was Click XION, the mobile phase A was 0.2% trifluoroacetic acid, and B was acetonitrile. The chromatographic conditions were 0-60 min, 100%-70% B, the injection volume was 50 g, and the flow rate was 80 mL / min. The high-performance liquid chromatography analysis and comparison were performed using Odyssil A C18 (250 × 4.6 mm, 5 μm) column was used, with a detection wavelength of 300 nm. Mobile phase A consisted of 0.2% trifluoroacetic acid in water, and mobile phase B consisted of acetonitrile. Mobile phase B was adjusted from 0 to 30 min, with a flow rate of 1.0 mL / min. Step 4, preparation of a reversed-phase high-pressure preparative column: Component Fr5 was separated by a reversed-phase column and detected by an ultraviolet detector at a wavelength of 300 nm. The corresponding chromatographic peak component Fr5-1 in the preparative chromatogram was collected and analyzed by HPLC. The retention times of component Fr5-1 and component Fr5 were compared to confirm the active target component. 47.28 mg of the target component Fr5-1 was obtained by vacuum drying. The vacuum concentration conditions were 50 mbar and 40°C. The working parameters for the preparation of the reversed-phase high-pressure preparative column used for component Fr5 were as follows: the preparative column was 250 mm long and 20 mm in diameter. The stationary phase of the reversed-phase column was 5 μm pure water-resistant C18. The mobile phase A was 0.2% trifluoroacetic acid-water solution. The mobile phase B was methanol solution. The elution was performed at 15%-35% B over 0-45 min. The injection volume was 4 mL and the flow rate was 19 mL / min. The HPLC analysis and comparison were performed using an Odyssil C18 (250×4.6 The detection wavelength was 300 nm, and the mobile phase A was 0.2% trifluoroacetic acid-water solution. The mobile phase B was acetonitrile solution. The flow rate was 1.0 mL / min, with a flow rate of 5%-30% B from 0 to 30 min. Step 5, preparation of a hydrophilic high-pressure preparative column: Component Fr5-1 was separated by a hydrophilic chromatographic column and detected by an ultraviolet detector at a detection wavelength of 300 nm. The corresponding chromatographic peak components in the preparative chromatogram were collected and analyzed by high-performance liquid chromatography (HPLC). The retention times of component Fr5-1-1 and component Fr5-1 were compared to confirm the active target component. The target component Fr5-1-1 was obtained by vacuum drying, wherein the vacuum concentration conditions were 50 mbar and 40°C. The working parameters for the preparation of the hydrophilic high-pressure preparative column used for the Fr5-1 component were as follows: the preparative column was 250 mm long and 20 mm in diameter, the hydrophilic chromatographic column stationary phase was 5 μm HILIC-D hydrophilic packing, the mobile phase A was 0.2% trifluoroacetic acid-water solution, the mobile phase B was acetonitrile solution, the elution was 80% B from 0 to 40 min, the injection volume was 4 mL, and the flow rate was 19 mL / min. The high-performance liquid chromatography analysis and comparison were performed using an Odyssil C18 (250×4.6 mm, 5 The detection wavelength was 300 nm, the mobile phase A was 0.2% trifluoroacetic acid-water solution, the mobile phase B was acetonitrile solution, the flow rate was 1.0 mL / min, and the flow rate was 5%-30% B from 0 to 30 min. 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, and the sample was filtered through a 0.45 μm microporous filter membrane to obtain filtrate C. The filtrate C was desalted, deacidified, and small molecular impurities were removed by gel chromatography column. The filtrate C was detected by an ultraviolet detector with a detection wavelength of 300 nm. The main chromatographic peak components in the preparative chromatogram were collected and each component was dried under vacuum to obtain 12.34 mg of the target new compound with a purity greater than 95%, named Lyciruthephenylpropanoid H, whose chemical structure (see Appendix for details) Figure 19 ); wherein, the conditions for vacuum concentration were a vacuum degree of 50 mbar and a temperature of 40°C; the operating parameters of the gel chromatography column were a column length of 1200 mm and a diameter of 30 mm, the stationary phase of the gel chromatography column was HW-40C, the mobile phase B was methanol, the chromatographic conditions were 0-1200 min, 100% B, and a flow rate of 1 mL / min.

[0019] Example 2 A process for separating Lyciruthephenylpropanoid H, a novel tyrosinase inhibitor from red wolfberry, comprises the following steps: Step 1, extraction: 1000 g of red wolfberry was dried in the shade, coarsely chopped, and extracted with 100% ethanol at a material-liquid ratio of 1 g:15 mL. The extraction was performed under vacuum heating and reflux for 3 times, each time for 3 hours. The filtrates were filtered, combined, and concentrated under vacuum to obtain a concentrate A. The vacuum concentration conditions were 150 mbar and 50°C. Step 2, online identification of tyrosinase binder components: 8 times the mass of methanol was added to the total sample of red wolfberry to dissolve it, the volume concentration of methanol was 80%, and the sample concentration was 80.0 mg / mL. The sample was filtered through a 0.45 μm microporous filter membrane to obtain a red wolfberry methanol sample solution, i.e., filtrate B; 1 mL of filtrate B was taken and used to identify the tyrosinase inhibitor containing the target component in red wolfberry using an online HPLC-FLD chromatography system; wherein, the online HPLC-FLD chromatography system, the first part of which is a high performance liquid chromatograph, using an Odyssil C18 (250×4.6 mm, 5 μm) column, and a detection wavelength of 300 nm; the second part is a fluorescence detector and a high performance liquid chromatography pump, the excitation wavelength and emission wavelength of the fluorescence detector are 221 nm and 331 nm, respectively, the concentration of the tyrosinase aqueous solution pumped into the high performance liquid chromatography pump is 15 μg / mL, and the flow rate of the mobile phase is 1 mL / min; the mobile phase A used in the high performance liquid chromatography was 0.2% trifluoroacetic acid-water solution, and the mobile phase B was acetonitrile solution. The flow rate of the mobile phase was 1.0 mL / min according to the flow rate of 5%-30% B from 0 to 30 min. Step 3, hydrophilic preparative column crude fractionation: filtrate B was wet loaded and injected into a medium-pressure chromatographic column filled with hydrophilic filler, and the sample was separated by the chromatographic column. During the separation process, the effluent was monitored by a UV detector with a wavelength of 300 nm. According to the detection results, the target chromatographic peak components in the preparative chromatogram were collected and analyzed by high-performance liquid chromatography (HPLC). The retention time of component Fr5 was compared with that of the total red wolfberry sample to confirm the active target component. After vacuum decompression and drying, 3.44 g of the target component Fr5 was obtained. The vacuum decompression and concentration conditions were a vacuum degree of 150 mbar and a temperature of 50°C. The operating parameters of the hydrophilic preparative column separation were as follows: the column length was 460 mm and the diameter was 49 mm, the stationary phase was Click XION, the mobile phase A was 0.2% trifluoroacetic acid, and B was acetonitrile. The chromatographic conditions were 0-60 min, 100%-70% B, the injection volume was 50 g, and the flow rate was 80 mL / min. The high-performance liquid chromatography analysis and comparison were performed using Odyssil A C18 (250 × 4.6 mm, 5 μm) column was used, with a detection wavelength of 300 nm. Mobile phase A consisted of 0.2% trifluoroacetic acid in water, and mobile phase B consisted of acetonitrile. Mobile phase B was adjusted from 0 to 30 min, with a flow rate of 1.0 mL / min. Step 4, preparation of a reversed-phase high-pressure preparative column: Component Fr5 was separated by a reversed-phase column and detected by a UV detector at a wavelength of 300 nm. The corresponding chromatographic peak component Fr5-1 in the preparative chromatogram was collected and analyzed by HPLC. The retention times of component Fr5-1 and component Fr5 were compared to confirm the active target component. 100.32 mg of the target component Fr5-1 was obtained by vacuum drying. The vacuum concentration conditions were 150 mbar and 50°C. The working parameters for the preparation of the reversed-phase high-pressure preparative column used for component Fr5 were as follows: the preparative column was 250 mm long and 20 mm in diameter. The stationary phase of the reversed-phase column was 5 μm pure water-resistant C18. The mobile phase A was 0.2% trifluoroacetic acid-water solution, and the mobile phase B was methanol solution. The elution was performed at 15%-35% B over 0-45 min. The injection volume was 4 mL and the flow rate was 19 mL / min. The HPLC analysis and comparison were performed using an Odyssil C18 (250×4.6 The detection wavelength was 300 nm, and the mobile phase A was 0.2% trifluoroacetic acid-water solution. The mobile phase B was acetonitrile solution. The flow rate was 1.0 mL / min, with a flow rate of 5%-30% B from 0 to 30 min. Step 5, preparation of a hydrophilic high-pressure preparative column: Component Fr5-1 was separated by a hydrophilic chromatographic column and detected by an ultraviolet detector at a wavelength of 300 nm. The corresponding chromatographic peak components in the preparative chromatogram were collected and analyzed by high-performance liquid chromatography (HPLC). The retention times of component Fr5-1-1 and component Fr5-1 were compared to confirm the active target component. The target component Fr5-1-1 was obtained by vacuum drying, wherein the vacuum concentration conditions were 150 mbar and 50°C. The working parameters for the preparation of the hydrophilic high-pressure preparative column used for the Fr5-1 component were as follows: the preparative column was 250 mm long and 20 mm in diameter, the hydrophilic chromatographic column stationary phase was 5 μm HILIC-D hydrophilic packing, the mobile phase A was 0.2% trifluoroacetic acid-water solution, the mobile phase B was acetonitrile solution, the elution was 80% B from 0 to 40 min, the injection volume was 4 mL, and the flow rate was 19 mL / min. The high-performance liquid chromatography analysis and comparison were performed using an Odyssil C18 (250×4.6 mm, 5 The detection wavelength was 300 nm, the mobile phase A was 0.2% trifluoroacetic acid-water solution, the mobile phase B was acetonitrile solution, the flow rate was 1.0 mL / min, and the flow rate was 5%-30% B from 0 to 30 min. 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, and filtered through a 0.45 μm microporous filter membrane to obtain filtrate C. Filtrate C was desalted, deacidified, and small molecular impurities were removed by gel chromatography column. The main chromatographic peak components in the preparative chromatogram were collected and each component was dried under vacuum to obtain 28.66 mg of the target new compound with a purity greater than 95%, named Lyciruthephenylpropanoid H, whose chemical structure (see Appendix for details) Figure 19 ); wherein, the conditions for vacuum concentration were a vacuum degree of 150 mbar and a temperature of 50°C; the operating parameters of the gel chromatography column were a column length of 1200 mm and a diameter of 30 mm, the stationary phase of the gel chromatography column was HW-40C, the mobile phase B was methanol, the chromatographic conditions were 0-1200 min, 100% B, and a flow rate of 1 mL / min.

[0020] Example 3 A process for separating Lyciruthephenylpropanoid H, a novel tyrosinase inhibitor from red wolfberry, comprises the following steps: Step 1, extraction: 5000 g of red wolfberry was dried in the shade, coarsely chopped, and extracted with 100% ethanol at a material-liquid ratio of 1 g:20 mL. The extraction was performed twice under vacuum heating and reflux, each for 4 hours. The filtrates were filtered, combined, and concentrated under vacuum to obtain a concentrate A. The vacuum concentration conditions were 250 mbar and 60°C. Step 2, online tyrosinase binder component identification: add 10 times the weight of methanol to the total red wolfberry sample to dissolve it, the volume concentration of methanol is 70%, and the sample concentration is prepared to be 50.0 mg / mL. Filter through a 0.45 μm microporous filter membrane to obtain the red wolfberry methanol sample solution, i.e., filtrate B; take 1 mL of filtrate B, and use the online HPLC-FLD chromatography system to identify the target component tyrosinase inhibitor (attached) in red wolfberry. Figure 1As shown); wherein, the online HPLC-FLD chromatography coupling system, the first part is a high performance liquid chromatograph, using an Odyssil C18 (250×4.6 mm, 5 μm) chromatographic column, and the detection wavelength is 300 nm; the second part is a fluorescence detector and a high performance liquid chromatography pump, the excitation wavelength and emission wavelength of the fluorescence detector are 221 nm and 331 nm, respectively, the concentration of tyrosinase aqueous solution pumped by the high performance liquid chromatography pump is 15 μg / mL, and the mobile phase flow rate is 1 mL / min; wherein, the mobile phase A used in 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, and the mobile phase flow rate is 1.0 mL / min; Step 3, hydrophilic medium pressure preparative column crude fractionation: filtrate B was wet loaded and injected into a medium pressure chromatographic column filled with hydrophilic filler. The sample was separated by the chromatographic column. During the separation process, the effluent was monitored by a UV detector with a wavelength of 300 nm. According to the detection results, the target chromatographic peak components in the preparative chromatogram were collected (see Appendix). Figure 2 By HPLC analysis, the retention time of component Fr5 was compared with that of the total sample of red wolfberry to confirm the active target component (see Figure 3 As shown), after vacuum drying, 16.32 g of the target component Fr5 was obtained. The vacuum concentration conditions were 250 mbar and 60°C. The hydrophilic preparative column separation parameters were as follows: a 460 mm long column with a diameter of 49 mm, Click XION stationary phase, 0.2% trifluoroacetic acid as mobile phase A, and acetonitrile as mobile phase B. The chromatographic conditions were 0-60 min, 100%-70% B, an injection volume of 50 g, and a flow rate of 80 mL / min. HPLC analysis and comparison were performed using an Odyssil C18 (250×4.6 mm, 5 μm) column with a detection wavelength of 300 nm. The mobile phases A and B were 0.2% trifluoroacetic acid-water solution, 5%-30% B, and 1.0 mL / min as mobile phase B. Step 4, preparation of reverse phase high pressure preparative column: Component Fr5 was separated by reverse phase chromatography column and detected by UV detector with a detection wavelength of 300 nm. The corresponding chromatographic peak component Fr5-1 in the preparative chromatogram was collected and analyzed by HPLC. The retention time of component Fr5-1 was compared with that of component Fr5 (see Appendix). Figure 5 The active target component was confirmed and 602.33 mg of the target component Fr5-1 (attached) was obtained by vacuum drying. Figure 4As shown), the vacuum concentration conditions were 250 mbar and 60°C. The working parameters for the preparation of the reversed-phase high-pressure preparative column used for component Fr5 were as follows: the preparative column was 250 mm long and 20 mm in diameter, the stationary phase of the reversed-phase column was 5 μm pure water-resistant C18, the mobile phase A was 0.2% trifluoroacetic acid-water, the mobile phase B was methanol solution, the elution was 15%-35% B over 0-45 min, the injection volume was 4 mL, and the flow rate was 19 mL / min. The HPLC analysis and comparison were performed using an Odyssil C18 (250×4.6 mm, 5 μm) column with a detection wavelength of 300 nm. The mobile phase A was 0.2% trifluoroacetic acid-water, the mobile phase B was acetonitrile solution, the elution was 5%-30% B over 0-30 min, and the mobile phase flow rate was 1.0 mL / min. Step 5, preparation of hydrophilic high pressure preparative column: Component Fr5-1 was separated by hydrophilic chromatographic column, detected by UV detector with a detection wavelength of 300nm, and the corresponding chromatographic peak components in the preparative chromatogram were collected (see Appendix Figure 6 The active target component was confirmed by HPLC analysis, and the target component Fr5-1-1 was obtained by vacuum drying. The vacuum concentration conditions were 250 mbar and 60°C. The working parameters of the hydrophilic high-pressure preparative column used for the Fr5-1 component were as follows: the preparative column was 250 mm long and 20 mm in diameter, the stationary phase of the hydrophilic column was 5 μm HILIC-D hydrophilic packing, the mobile phase A was 0.2% trifluoroacetic acid-water, the mobile phase B was acetonitrile, the elution time was 0-40 min, the injection volume was 4 mL, and the flow rate was 19 mL / min. The HPLC analysis and comparison were performed using an Odyssil C18 (250×4.6 mm, 5 μm) column with a detection wavelength of 300 nm, a mobile phase A was 0.2% trifluoroacetic acid-water, and a mobile phase B was acetonitrile. The elution time was 0-30 min, the injection volume was 4 mL, and the flow rate was 19 mL / min. min, 5%-30% B, mobile phase flow rate was 1.0 mL / min; 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, and the mixture was filtered through a 0.45 μm microporous filter membrane to obtain filtrate C. The filtrate C was desalted, deacidified, and subjected to a gel chromatography column to remove small molecule impurities. The filtrate C was detected by an ultraviolet detector with a detection wavelength of 300 nm, and the main chromatographic peak components in the preparative chromatogram were collected (see Appendix). Figure 7), each component was dried under vacuum to obtain 150.86 mg of the target new compound with a purity greater than 95%, named Lyciruthephenylpropanoid H, and its chemical structure (see Appendix for details) Figure 19 ); wherein, the conditions for vacuum decompression concentration are a vacuum degree of 250 mbar and a temperature of 60°C; the operating parameters of the gel chromatography column are a column length of 1200 mm and a diameter of 30 mm, the stationary phase of the gel chromatography column is HW-40C, the mobile phase B is methanol, the chromatographic conditions are 0-1200 min, 100% B, and a flow rate of 1 mL / min.

[0021] Example 4 Activity verification of Lyciruthephenylpropanoid H, a new structure tyrosinase inhibitor in red wolfberry: Lyciruthephenylpropanoid H, a novel tyrosinase inhibitor isolated from red wolfberry, was dissolved in 4 times its weight of chromatographic methanol to prepare a sample concentration of 0.2 mg / mL. The sample solution was filtered through a 0.45 μm microporous filter to obtain a new structure tyrosinase inhibitor sample solution from red wolfberry. 1 mL of the sample was taken and the activity of the novel structure tyrosinase inhibitor Lyciruthephenylpropanoid H from red wolfberry was verified using an online HPLC-FLD chromatography system. The online HPLC-FLD chromatography system comprises a high performance liquid chromatograph as the first part, which uses an Odyssil (250×4.6 mm, 5 μm) chromatographic column. The mobile phase A used by the first high performance liquid chromatograph is 0.2% trifluoroacetic acid-water solution, and the mobile phase B is acetonitrile solution. The mobile phase is 5-30% B in 0-30 min, the mobile phase flow rate is 1.0 mL / min, and the detection wavelength is 300 nm. The second part comprises a fluorescence detector and a high performance liquid chromatography pump. The excitation wavelength and emission wavelength of the fluorescence detector are 221 nm and 331 nm, respectively. The concentration of the tyrosinase aqueous solution pumped into the high performance liquid chromatography pump is 15 μg / mL, the mobile phase flow rate is 1.0 mL / min, and the activity verification chromatogram (attached) is shown in FIG. Figure 8 shown).

[0022] Therefore, the new structure tyrosinase inhibitor Lyciruthephenylpropanoid H isolated from the red wolfberry is used in the preparation of drugs or health foods that reduce melanin production. The new structure tyrosinase inhibitor Lyciruthephenylpropanoid H is used as an active ingredient and is prepared into various pharmaceutical preparations according to conventional methods with any pharmaceutically acceptable carrier, or is used as an active ingredient and is prepared into various health foods according to conventional methods with any carrier acceptable in food science.

[0023] Example 5 In vitro inhibition experiment of Lyciruthephenylpropanoid H, a new structure tyrosinase inhibitor in red wolfberry: 40 μL of sample solution of the target new compound diluted with PBS buffer at appropriate concentrations was added to the microplate; 20 μL of L-tyrosine solution prepared with 0.2 mg / mL PBS buffer and 30 μL of Tris-HCl buffer were added in sequence, so that the final reaction concentrations of the samples were 0.25, 0.50, 1.00, 2.00, 3.00 and 4.00 mmol / L, respectively. The samples were incubated at 37°C for 10 min, and 40 μL of tyrosinase solution prepared with 98.5 μg / mL PBS buffer was added. The samples were incubated at 37°C for 10 min, and the absorbance was measured at 475 nm. The half-maximal inhibitory concentration (IC) of different tyrosinase inhibitors was calculated using SPSS 19.0 software. 50 Kojic acid was used as the positive control. The blank group used an equal amount of PBS buffer to replace the sample solution. The blank control group used an equal amount of PBS buffer to replace the sample and enzyme solution. The sample control group used an equal amount of PBS buffer to replace the enzyme solution (see Appendix). Figure 9 The inhibition rates of the samples on tyrosinase are as follows: Where, ODs, ODs0, OD b ,OD b0 Represent the absorbance values ​​of the sample group, sample control group, blank group and blank control group, respectively.

[0024] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A process for separating Lyciruthephenylpropanoid H, a novel tyrosinase inhibitor from red wolfberry, characterized in that: The specific steps include: Step 1, extraction: Dry the red wolfberry in the shade, coarsely chop it, and extract it with 100% ethanol at a material-liquid ratio of 1 g: 10-20 mL. Heat under vacuum reflux for 2-4 times, each time for 2-4 hours. Filter, combine the filtrates, and concentrate the filtrates under vacuum to obtain concentrate A. Step 2, online tyrosinase inhibitor component identification: 5 to 10 times the mass of methanol was added to the total red wolfberry sample to dissolve it, the volume concentration of the methanol being 70% to 90%, and the sample concentration being prepared to be 50.0 to 100.0 mg / mL. The sample was filtered through a 0.45 μm microporous filter membrane to obtain a red wolfberry methanol sample solution, i.e., filtrate B. 1 mL of filtrate B was taken and used to identify the tyrosinase inhibitor containing the target component in the red wolfberry using an online HPLC-FLD chromatography system. The online HPLC-FLD chromatography system comprised a high performance liquid chromatograph, a first part using an Odyssil C18 (250×4.6 mm, 5 μm) column, and a detection wavelength of 300 nm. The second part comprised a fluorescence detector and a high performance liquid chromatography pump, the excitation wavelength and emission wavelength of the fluorescence detector being 221 nm and 331 nm, respectively, and the high performance liquid chromatography pump pumped the tyrosinase aqueous solution. Step 3, hydrophilic medium-pressure preparative column crude fractionation: Filtrate B is wet-loaded and injected into a medium-pressure chromatographic column filled with hydrophilic filler. The sample is separated using the chromatographic column. During the separation process, the effluent is monitored using a UV detector with a wavelength of 300 nm. Based on the detection results, the target chromatographic peak components in the preparative chromatogram are collected and analyzed by high-performance liquid chromatography (HPLC). The retention time of component Fr5 is compared with that of the total red wolfberry sample to confirm the active target component. After the component is dried under vacuum, the target component Fr5 can be obtained. Step 4, reverse-phase high-pressure preparative column preparation: The component Fr5 is separated by a reverse-phase chromatographic column and detected by an ultraviolet detector with a detection wavelength of 300 nm. The corresponding chromatographic peak component Fr5-1 in the preparative chromatogram is collected and analyzed by high-performance liquid chromatography (HPLC). The retention time of component Fr5-1 and component Fr5 is compared to confirm the active target component, and the target component Fr5-1 is obtained by vacuum drying. Step 5, preparation of a hydrophilic high-pressure preparative column: The component Fr5-1 is separated by a hydrophilic chromatographic column and detected by an ultraviolet detector with a detection wavelength of 300 nm. The corresponding chromatographic peak components in the preparative 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, and the sample was filtered through a 0.45 μm microporous filter membrane to obtain filtrate C. The filtrate C was desalted, deacidified, and small molecular impurities were removed by gel chromatography column. The filtrate C was detected by an ultraviolet detector with a detection wavelength of 300 nm. The main chromatographic peak components in the preparative chromatogram were collected and each component was dried under vacuum to obtain a target new compound with a purity greater than 95%, named Lyciruthephenylpropanoid H, whose chemical structure is as follows: ； Step 7, in vitro tyrosinase inhibition assay: 40 μL of a sample solution of the target new compound diluted with PBS buffer at appropriate concentrations was added to the microplate; 20 μL of a 0.2 mg / mL L-tyrosine solution prepared with PBS buffer and 30 μL of Tris-HCl buffer were added in sequence, so that the final reaction concentrations of the samples were 0.25, 0.50, 1.00, 2.00, 3.00 and 4.00 mmol / L, respectively. The samples were incubated at 37°C for 10 min, and 40 μL of a tyrosinase solution prepared with 98.5 μg / mL PBS buffer was added. The samples were incubated at 37°C for 10 min, and the absorbance was measured at 475 nm. The half-maximal inhibitory concentration (IC) of different tyrosinase inhibitors was calculated using SPSS 19.0 software. 50 Kojic acid was used as the positive control. The blank group used an equal amount of PBS buffer to replace the sample solution. The blank control group used an equal amount of PBS buffer to replace the sample and enzyme solution. The sample control group used an equal amount of PBS buffer to replace the enzyme solution. The inhibition rate of the sample on tyrosinase is as follows: Where, ODs, ODs0, OD b ,OD b0 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, wherein: In step 1, step 3, step 4, step 5, and step 6, the conditions for vacuum decompression concentration are: vacuum degree 50-250 mbar, temperature 40-60°C.

3. The separation process according to claim 1, wherein: In step 2, the mobile phase A used in 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, and the mobile phase flow rate is 1.0 mL / min; the tyrosinase concentration pumped into the high performance liquid chromatography pump is 15 μg / mL, and the mobile phase flow rate is 1 mL / min.

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

5. The separation process according to claim 1, wherein: In step 4, the working parameters for the preparation of the reversed-phase high-pressure preparative column used for the component Fr5 are as follows: the preparative column has a length of 250 mm and a diameter of 20 mm, the stationary phase of the reversed-phase chromatographic column is 5 μm pure water-resistant C18, the mobile phase A is 0.2% trifluoroacetic acid-water solution, the mobile phase B is methanol solution, according to 0~45 min, 15%-35% B elution, the injection volume is 4 mL, and the flow rate is 19 mL / min; for high performance liquid chromatography analysis and comparison, an Odyssil C18 (250×4.6 mm, 5 μm) column is used, the detection wavelength is 300 nm, the mobile phase A used is 0.2% trifluoroacetic acid-water solution, the mobile phase B is acetonitrile solution, according to 0~30 min, 5%-30% B, and the mobile phase flow rate is 1.0 mL / min.

6. The separation process according to claim 1, wherein: In step 5, the working parameters for the preparation of the hydrophilic high-pressure preparative column used for the Fr5-1 component are as follows: the preparative column is 250 mm long and 20 mm in diameter, the hydrophilic chromatographic column stationary phase 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, according to 0~40 min, 80% B elution, the injection volume is 4 mL, and the flow rate is 19 mL / min; for high performance liquid chromatography analysis and comparison, an Odyssil C18 (250×4.6 mm, 5 μm) column is used, the detection wavelength is 300 nm, the mobile phase A is 0.2% trifluoroacetic acid-water solution, the mobile phase B is acetonitrile solution, according to 0~30 min, 5%-30% B, and the mobile phase 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: a column length of 1200 mm and a diameter of 30 mm, a gel chromatography column stationary phase of HW-40C, a mobile phase B of methanol, a chromatographic condition of 0-1200 min, 100% B, and a flow rate of 1 mL / min.

8. A use of Lyciruthephenylpropanoid H, a novel tyrosinase inhibitor in red wolfberry, characterized by: The novel structure tyrosinase inhibitor Lyciruthephenylpropanoid H is prepared according to any one of claims 1 to 7. The tyrosinase inhibitor Lyciruthephenylpropanoid H is used in the preparation of a drug or health food for reducing melanin production. The novel structure tyrosinase inhibitor Lyciruthephenylpropanoid H is used as an active ingredient and is prepared into various pharmaceutical preparations according to conventional methods with any pharmaceutically acceptable carrier, or is used as an active ingredient and is prepared into various health foods according to conventional methods with any carrier acceptable in food science.

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