6 '-O-caffeoyl arbutin and extraction method thereof

Abstract

The invention provides 6 '-O-caffeoyl arbutin and an extraction method thereof, and belongs to the field of natural substance extraction. The extraction method of the 6 '-O-caffeoyl arbutin provided by the invention comprises the following steps: mixing an azaleaceae vaccinium plant with water, and sequentially carrying out heating reflux, vacuum concentration, alcohol precipitation, water precipitation, chromatographic purification, concentration and drying to obtain the 6'-O-caffeoyl arbutin. According to the method for extracting the 6 '-O-caffeoyl arbutin from the rhododenaceae vaccinium plants, the rhododenaceae vaccinium plants are subjected to multiple times of enrichment and impurity removal after being extracted, interference of other substances is effectively reduced, the yield of the extract and the purity of the 6'-O-caffeoyl arbutin are improved, the total extraction rate obtained through the extraction method ranges from 52.01% to 66.50%, and the purity of the 6 '-O-caffeoyl arbutin ranges from 98.12% to 98.56%.

Description

Technical Field

[0001] The invention relates to the field of natural medicine preparation, and in particular to 6'-O-caffeoyl arbutin and an extraction method thereof. Background Art

[0002] 6'-O-caffeoyl arbutin is an arbutin derivative and a compound with multiple biological activities. It has excellent antioxidant, anti-inflammatory and antibacterial effects, showing potential benefits to human health and attracting attention in natural product research. The compound was originally extracted from the leaves of Betula leucoderma or Hakea plants of the Proteaceae family, and is now mostly extracted from Vaccinium plants of the Ericaceae family. However, in the prior art, the extraction of 6'-O-caffeoyl arbutin generally has the problems of low extraction rate and low content of effective substances, i.e., low purity.

[0003] For example, the prior art CN116898778A discloses a method for extracting 6'-O-caffeoyl arbutin from the terminal buds or tender leaves of Vaccinium camphorum, a plant of the genus Vaccinium of the Ericaceae family. The yield of the Vaccinium camphorum extract obtained by this method is 10.36-11.20%, and the content of 6'-O-caffeoyl arbutin in the Vaccinium camphorum extract is 72.83%-75.92%; CN111471078A discloses a method for extracting 6'-O-caffeoyl arbutin using Vaccinium camphorum as a raw material. This method can only obtain 20.4g of product from 5kg of Vaccinium camphorum raw material, and the extraction rate is extremely low; CN113 332194A discloses a method for extracting 6'-O-caffeoyl arbutin from quezui tea by using microwave-subcritical water extraction. In this method, the temperature required for subcritical water extraction is high, which easily destroys the structure of the effective ingredient 6'-O-caffeoyl arbutin, and the extraction rate of quezui tea extract is 16-26%, and the content of 6'-O-caffeoyl arbutin in the quezui tea extract is 72.5-73.56%; CN111035669A discloses a method for extracting 6'-O-caffeoyl arbutin from quezui tea. The content of 6'-O-caffeoyl arbutin in the quezui tea extract obtained by this method is above 60%.

[0004] In summary, there are many methods for extracting 6'-O-caffeoyl arbutin from Vaccinium vulgare of Ericaceae in the prior art, but the yield of Vaccinium vulgare extracts obtained by these methods is low, and the purity of 6'-O-caffeoyl arbutin is also low, and the extraction rate and purity cannot meet the needs of practical applications. Summary of the invention

[0005] The purpose of the present invention is to provide a 6'-O-caffeoyl arbutin and an extraction method thereof. The extraction method provided by the present invention can effectively improve the extraction rate and the purity of 6'-O-caffeoyl arbutin.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for extracting 6'-O-caffeoyl arbutin, comprising the following steps:

[0008] (1) mixing a plant of the genus Vaccinium in the family Ericaceae with water and heating the mixture under reflux to obtain an extract;

[0009] (2) concentrating the extract obtained in step (1) under reduced pressure to obtain an extract concentrate;

[0010] (3) subjecting the extract concentrate obtained in step (2) to alcohol precipitation to obtain an alcohol precipitate;

[0011] (4) subjecting the alcohol precipitation liquid obtained in step (3) to water precipitation to obtain a water precipitation liquid;

[0012] (5) chromatographically purifying the water precipitate obtained in step (4) to obtain an eluent;

[0013] (6) The eluate obtained in step (5) is concentrated and then crystallized to obtain 6'-O-caffeoyl arbutin.

[0014] Preferably, the temperature of heating and reflux in step (1) is 90 to 110° C., the number of heating and reflux is 2 to 5 times, and the time of each heating and reflux is 1 to 3 hours.

[0015] Preferably, the temperature of the reduced pressure concentration in step (2) is 40 to 70° C., the pressure of the reduced pressure concentration is -0.06 to -0.1 MPa, and the endpoint of the reduced pressure concentration is that the relative density of the extract concentrate is 1.10 to 1.25 g / mL.

[0016] Preferably, the reagent for alcohol precipitation in step (3) is an ethanol solution, the temperature of alcohol precipitation is 2 to 25° C., and the time of alcohol precipitation is 8 to 24 hours.

[0017] Preferably, the mass concentration of the ethanol solution is 60-95%, and the volume ratio of the extraction concentrate to the ethanol solution is 1:(2.5-10).

[0018] Preferably, the water precipitation temperature in step (4) is room temperature, and the water precipitation time is 8 to 24 hours.

[0019] Preferably, the chromatographic column filler for chromatographic purification in step (5) is a reverse phase C18 chromatographic column filler or a reverse phase polymer chromatographic column filler, the elution solvent for chromatographic purification is an ethanol solution, the detection wavelength for chromatographic purification is 326 nm, the loading amount for chromatographic purification is 3 to 10 g / L, and the time for chromatographic purification is from the beginning of the chromatogram corresponding to 6'-O-caffeoyl arbutin to the end of the chromatogram corresponding to 6'-O-caffeoyl arbutin.

[0020] Preferably, the mass concentration of the ethanol solution is 30-35%, and the flow rate of the ethanol solution is 10-30 min / BV.

[0021] Preferably, the concentration pressure in step (6) is -0.06 to -0.1 MPa, the concentration temperature is 40 to 70°C, and the concentration endpoint is when the volume of the concentrate is 0.035 to 0.1 times the volume of the eluent.

[0022] Preferably, the crystallization temperature in step (6) is 2 to 8° C., and the crystallization time is 8 to 18 hours.

[0023] The invention provides a method for extracting 6'-O-caffeoyl arbutin, comprising the following steps: (1) mixing a plant of the genus Vaccinium of the Ericaceae family with water and heating and refluxing the mixture to obtain an extract; (2) concentrating the extract obtained in the step (1) under reduced pressure to obtain an extract concentrate; (3) subjecting the extract concentrate obtained in the step (2) to alcohol precipitation to obtain an alcohol precipitation liquid; (4) subjecting the alcohol precipitation liquid obtained in the step (3) to water precipitation to obtain a water precipitation liquid; (5) subjecting the water precipitation liquid obtained in the step (4) to chromatography purification to obtain an eluent; and (6) concentrating the eluent obtained in the step (5) and crystallizing the mixture to obtain 6'-O-caffeoyl arbutin. The invention dynamically extracts the plant of Ericaceae Vaccinium by heating and refluxing, thereby increasing the driving force of leaching and improving the extraction rate; by reducing pressure and concentrating, effective enrichment of 6'-O-caffeoyl arbutin can be achieved, the extraction rate is further improved, and the purity of 6'-O-caffeoyl arbutin is improved; by alcohol precipitation and then water precipitation of the concentrated solution, the enrichment degree of 6'-O-caffeoyl arbutin can be further improved, and the impurities in the concentrated solution can be eluted at the same time, thereby further improving the purity of 6'-O-caffeoyl arbutin; by chromatographic purification, fine purification of 6'-O-caffeoyl arbutin can be achieved, and the purity of 6'-O-caffeoyl arbutin can be greatly improved; by the post-treatment operation of concentration and drying, the enrichment and impurity removal of 6'-O-caffeoyl arbutin can be achieved again, and the extraction rate of the extract and the purity of 6'-O-caffeoyl arbutin can be further improved. The data in the examples show that the total extraction rate obtained by the extraction method provided by the present invention is 52.01-66.50%, and the purity of 6'-O-caffeoyl arbutin is 98.12-98.56%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the HPLC test results of 6'-O-caffeoyl arbutin extracted in Example 1 of the present invention;

[0025] Figure 2 This is the H NMR spectrum of 6'-O-caffeoyl arbutin extracted in Example 1 of the present invention;

[0026] Figure 3 This is the C NMR spectrum of 6'-O-caffeoyl arbutin extracted in Example 1 of the present invention;

[0027] Figure 4 This is a graph showing the HPLC test result of 6'-O-caffeoyl arbutin extracted in Comparative Example 1 of the present invention;

[0028] Figure 5 This is a graph showing the HPLC test result of 6'-O-caffeoyl arbutin extracted in Comparative Example 2 of the present invention;

[0029] Figure 6 This is a graph showing the HPLC detection results of 6'-O-caffeoyl arbutin extracted in Comparative Example 3 of the present invention. DETAILED DESCRIPTION

[0030] The present invention provides a method for extracting 6'-O-caffeoyl arbutin, comprising the following steps:

[0031] (1) mixing a plant of the genus Vaccinium in the family Ericaceae with water and heating the mixture under reflux to obtain an extract;

[0032] (2) concentrating the extract obtained in step (1) under reduced pressure to obtain an extract concentrate;

[0033] (3) subjecting the extract concentrate obtained in step (2) to alcohol precipitation to obtain an alcohol precipitate;

[0034] (4) subjecting the alcohol precipitation liquid obtained in step (3) to water precipitation to obtain a water precipitation liquid;

[0035] (5) chromatographically purifying the water precipitate obtained in step (4) to obtain an eluent;

[0036] (6) The eluate obtained in step (5) is concentrated and then crystallized to obtain 6'-O-caffeoyl arbutin.

[0037] The invention mixes the plant of Ericaceae genus Vaccinium with water and heats and refluxes the mixture to obtain an extract.

[0038] The present invention has no special limitation on the source of the plant of the genus Vaccinium of Ericaceae, and conventional commercial products can be used.

[0039] In the present invention, the plant of the genus Vaccinium in the family Ericaceae is preferably Vaccinium grandiflorum.

[0040] In the present invention, the particle size of the plant of the genus Vaccinium of Ericaceae is preferably 24-65 meshes, more preferably 30-55 meshes, and most preferably 40-50 meshes. In the present invention, by limiting the particle size of the raw material, effective extraction of 6'-O-caffeoyl arbutin can be achieved.

[0041] In the present invention, the mass ratio of the Ericaceae Vaccinium plant to water is preferably 1:(5-15), more preferably 1:(7-11), and most preferably 1:(8-10). In the present invention, by limiting the usage ratio of the Ericaceae Vaccinium plant to water, the dissolution effect of the Ericaceae Vaccinium plant and the extraction effect of 6'-O-caffeoyl arbutin in the Ericaceae Vaccinium plant are further improved.

[0042] In the present invention, the temperature of the heating reflux is preferably 90-110°C, more preferably 95-105°C, and most preferably 98-102°C; the number of the heating reflux is preferably 2-5 times, more preferably 3-4 times; the time of each heating reflux is preferably 1-3 hours, more preferably 1.5-2.5 hours, and most preferably 1.8-2 hours. In the present invention, by limiting the process parameters of heating reflux, the leaching driving force is increased, the dynamic extraction effect of 6'-O-caffeoyl arbutin is improved, and the extraction rate is further improved.

[0043] In the present invention, it is preferred that solutions obtained by heating and refluxing for multiple times are combined to obtain an extract.

[0044] After obtaining the extract, the present invention performs reduced pressure concentration on the extract to obtain an extract concentrate.

[0045] In the present invention, the temperature of the reduced pressure concentration is preferably 40 to 70°C, more preferably 45 to 65°C, and most preferably 50 to 60°C; the pressure of the reduced pressure concentration is preferably -0.06 to -0.1MPa, more preferably -0.07 to -0.09MPa, and most preferably -0.075 to -0.08MPa; the endpoint of the reduced pressure concentration is preferably that the relative density of the extract concentrate is 1.10 to 1.25g / mL, more preferably 1.15 to 1.22g / mL, and most preferably 1.18 to 1.20g / mL. In the present invention, by limiting the process parameters of reduced pressure concentration, the effective enrichment of 6'-O-caffeoyl arbutin is achieved, and the extraction rate is further improved; and impurities can be removed while enriching, further improving the purity of 6'-O-caffeoyl arbutin.

[0046] After obtaining the extraction concentrated solution, the present invention performs alcohol precipitation on the extraction concentrated solution to obtain an alcohol precipitation solution.

[0047] In the present invention, the reagent for alcohol precipitation is preferably an ethanol solution; the concentration of the ethanol solution is preferably 60-95%, more preferably 65-85%, and most preferably 70-80%; the volume ratio of the extract concentrate to the ethanol solution is preferably 1:(2.5-10), more preferably 1:(4-9), and most preferably 1:(5-7); the temperature of the alcohol precipitation is preferably 2-25°C, more preferably 3-15°C, and most preferably 5-8°C; the time of the alcohol precipitation is preferably 8-24h, more preferably 10-20h, and most preferably 12-16h. In the present invention, by limiting the process parameters of alcohol precipitation, the enrichment degree of 6'-O-caffeoyl arbutin can be further improved, thereby further improving the purity of 6'-O-caffeoyl arbutin.

[0048] In the present invention, it is preferred that the alcohol precipitation reagent is added while stirring, and after the alcohol precipitation reagent is completely added, stirring is stopped and the mixture is allowed to stand for alcohol precipitation.

[0049] In the present invention, the stirring rate is preferably 10 to 50 rpm / min, more preferably 15 to 40 rpm / min, and most preferably 20 to 30 rpm / min. In the present invention, the enrichment effect of the alcohol precipitation treatment is further improved by limiting the stirring rate of adding the ethanol solution.

[0050] In the present invention, the alcohol precipitation is preferably followed by suction filtration, the filter residue obtained by suction filtration is washed with an ethanol solution, and the washing liquid is combined with the filtrate obtained by suction filtration to obtain an alcohol precipitation liquid.

[0051] The present invention has no special limitation on the suction filtration, and the suction filtration operation commonly used by those skilled in the art can be adopted.

[0052] In the present invention, the mass concentration of the ethanol solution used for washing is preferably 60-95%, more preferably 65-90%, and most preferably 70-80%; the number of washings is preferably 2-4, and more preferably 2-3. In the present invention, by limiting the concentration of the washing solution and the number of washings, the residual 6'-O-caffeoyl arbutin in the filter residue can be completely washed out, further improving the purity of the product.

[0053] After obtaining the alcohol precipitation liquid, the present invention performs water precipitation on the alcohol precipitation liquid to obtain the water precipitation liquid.

[0054] In the present invention, the alcohol precipitation liquid is preferably concentrated under reduced pressure and cooled naturally before water precipitation.

[0055] In the present invention, the temperature of the reduced pressure concentration is preferably 40 to 70°C, more preferably 45 to 65°C, and most preferably 50 to 60°C; the pressure of the reduced pressure concentration is preferably -0.06 to -0.1MPa, more preferably -0.07 to -0.09MPa, and most preferably -0.075 to -0.08MPa; the endpoint of the reduced pressure concentration is preferably a relative density of the alcohol precipitation liquid of 1.10 to 1.25g / mL, more preferably 1.15 to 1.22g / mL, and most preferably 1.18 to 1.20g / mL. In the present invention, by limiting the conditions of reduced pressure concentration, the 6'-O-caffeoyl arbutin obtained by alcohol precipitation is further concentrated, laying the foundation for subsequent water precipitation.

[0056] In the present invention, the water precipitation temperature is preferably room temperature; the water precipitation time is preferably 8 to 24 hours, more preferably 10 to 20 hours, and most preferably 14 to 16 hours. In the present invention, by limiting the water precipitation time, it is ensured that complete water precipitation can be achieved, and impurities in the alcohol precipitation liquid are eluted to the greatest extent, thereby further improving the purity of the product.

[0057] The present invention preferably performs stirring while water sinking, and the stirring rate is preferably 10 to 50 rpm / min, more preferably 15 to 40 rpm / min, and most preferably 20 to 30 rpm / min. In the present invention, the water sinking effect is further improved by limiting the stirring rate.

[0058] After obtaining the water precipitate, the present invention performs chromatography purification on the water precipitate to obtain an eluent.

[0059] The present invention preferably performs suction filtration on the water precipitate before chromatographic purification, washes the suction filtration residue with purified water to obtain a solid wet material, dissolves the solid wet material with an ethanol solution, filters it, and performs chromatographic purification on the filtrate.

[0060] The present invention has no special limitation on the suction filtration, and the suction filtration operation commonly used by those skilled in the art can be adopted; the present invention has no special limitation on the operation of washing with purified water, and the suction filtration residue can be washed until it is colorless.

[0061] In the present invention, the mass concentration of the ethanol solution for dissolving the solid wet material is preferably 55-65%, more preferably 58-62%, and most preferably 60%; the mass ratio of the solid wet material to the ethanol solution is preferably 1:(5-10), more preferably 1:(6-9), and most preferably 1:(7-8). In the present invention, by limiting the parameters of the ethanol solution for dissolving the solid wet material, it is ensured that the solid wet material can be dissolved to the greatest extent, the transfer of 6'-O-caffeoyl arbutin is achieved and the dissolution of other impurities is avoided, thereby further improving its purity.

[0062] In the present invention, the chromatographic column filler for chromatographic purification is preferably a reverse phase C18 chromatographic column filler or a reverse phase polymer chromatographic column filler, more preferably a reverse phase polymer chromatographic column filler, further preferably a Uni reverse phase polymer chromatographic column filler, and most preferably UniPS 30-100, UniPS 30-300, UniPS 40-300, UniPS10-300, UniPSN 30-300, UniPSN 40-300 or UniPMM40-500; the elution solvent of the chromatographic purification is preferably an ethanol solution; the mass concentration of the ethanol solution is preferably 30-35%, more preferably 31-34%, and most preferably 32-33%; the flow rate of the ethanol solution is preferably 10-30 min / BV, more preferably 15-25 min / BV, and most preferably 18-20 min / BV; the detection wavelength of the chromatographic purification is preferably 326 nm; the loading amount of the chromatographic purification is preferably 3-10 g / L, more preferably 4-8 g / L, and most preferably 5-6 g / L; the time of the chromatographic purification is preferably the starting point of the chromatogram corresponding to 6'-O-caffeoyl arbutin, and the end point is the end of the chromatogram corresponding to 6'-O-caffeoyl arbutin. In the present invention, by limiting the conditions of chromatographic purification, the effect of chromatographic purification is further improved, thereby further improving the purity of 6'-O-caffeoyl arbutin.

[0063] After obtaining the eluate, the present invention concentrates the eluate and then crystallizes it to obtain 6'-O-caffeoyl arbutin.

[0064] In the present invention, the concentration is preferably reduced pressure concentration; the concentration pressure is preferably -0.06 to -0.1 MPa, more preferably -0.07 to -0.09 MPa, and most preferably -0.075 to -0.08 MPa; the concentration temperature is preferably 40 to 70°C, more preferably 45 to 65°C, and most preferably 50 to 60°C; the concentration end point is preferably that the volume of the concentrate is 0.035 to 0.1 times the volume of the eluent, more preferably 0.05 to 0.09 times, and most preferably 0.06 to 0.08 times. In the present invention, by limiting the process parameters of the concentration operation, the enrichment of 6'-O-caffeoyl arbutin is further achieved.

[0065] In the present invention, crystallization is preferably static crystallization; the temperature of the static crystallization is preferably 2 to 8°C, more preferably 3 to 6°C, and most preferably 4 to 5°C; the time of the static crystallization is preferably 8 to 18 hours, more preferably 10 to 16 hours, and most preferably 12 to 14 hours. In the present invention, by limiting the crystallization conditions, the formation of crystals is promoted, and separation from impurities is achieved, thereby further improving the purity of the product.

[0066] After the crystallization is completed, the present invention preferably performs solid-liquid separation on the crystallized product in sequence, and washes and dries the obtained solid to obtain 6'-O-caffeoyl arbutin.

[0067] In the present invention, the solid-liquid separation is preferably suction filtration. The present invention has no special limitation on the suction filtration, and the suction filtration operation commonly used by those skilled in the art can be adopted. The washing operation of the present invention is not particularly limited, and the washing operation commonly used by those skilled in the art can be adopted.

[0068] In the present invention, the drying is preferably reduced-pressure drying; the drying time is preferably 1 to 5 hours, more preferably 2 to 4 hours, and most preferably 2.5 to 3 hours; the drying temperature is preferably 50 to 90°C, more preferably 60 to 80°C, and most preferably 65 to 70°C; the drying vacuum is preferably 5 to 20 bar, more preferably 8 to 16 bar, and most preferably 10 to 15 bar.

[0069] The extraction method provided by the invention realizes multiple extraction and impurity removal of Ericaceae Vaccinium plants, effectively improves the extraction rate of the extract and the purity of 6'-O-caffeoyl arbutin; and adopts a water extraction process for Ericaceae Vaccinium plant raw materials, thereby reducing the leaching of pigment substances, making decolorization easy and the finished product having excellent color; the overall process adopts an ethanol-water system, without the use of other organic solvents, thereby reducing the risk of solvent residues.

[0070] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0071] Example 1

[0072] A method for extracting 6'-O-caffeoyl arbutin comprises the following steps:

[0073] (1) Mixing 30-mesh quezui tea with water and heating and refluxing for 3 times at 110° C., each time for 1 hour, to obtain an extract; wherein the mass ratio of quezui tea to water is 1:11;

[0074] (2) the extract obtained in step (1) is concentrated under reduced pressure at 60° C. and −0.08 MPa to obtain an extract concentrate having a relative density of 1.15 g / mL;

[0075] (3) slowly adding an ethanol solution with a mass concentration of 95% to the extract concentrate obtained in step (2) at a stirring rate of 30 rpm / min, wherein the volume ratio of the extract concentrate to the ethanol solution is 1:2.8; after the ethanol solution is completely added, stopping stirring, standing at 5° C. for 12 h for alcohol precipitation, filtering, and washing the obtained suction filtration residue twice with an ethanol solution with a mass concentration of 95%, combining the washing liquid and the suction filtration filtrate to obtain an alcohol precipitate;

[0076] (4) the alcohol precipitate obtained in step (3) was concentrated under reduced pressure at 50° C. and −0.07 MPa to a relative density of 1.17 g / mL, cooled naturally, and then subjected to water precipitation for 12 h at a stirring rate of 20 rpm / min to obtain a water precipitate;

[0077] (5) filtering the water precipitate obtained in step (4), washing the filtered residue with purified water to obtain a solid wet material, dissolving the solid wet material with an ethanol solution with a mass concentration of 60%, wherein the mass ratio of the solid wet material to the ethanol solution is 1:7; filtering, and chromatographically purifying the filtrate to obtain an eluent; wherein the conditions for chromatographic purification are: the chromatographic column filler is a UniPSN 40-300 reverse phase polymer chromatographic column filler, the elution solvent is an ethanol solution with a mass concentration of 35%, the flow rate of the elution solvent is 15 min / BV, the detection wavelength is 326 nm, and the sample load is 5.7 g / L;

[0078] (6) The eluate obtained in step (5) was concentrated under reduced pressure at 50° C. and −0.08 MPa until the volume of the concentrate was 0.06 times the volume of the eluate, and the concentrate was allowed to stand at 5° C. for 12 h for crystallization. The crystals were filtered and washed with water, and then dried in a vacuum drying oven at 90° C. and 10 bar for 4 h to obtain 459.1 g of 6′-O-caffeoyl arbutin.

[0079] The 6'-O-caffeoyl arbutin obtained in Example 1 was detected by high performance liquid chromatography, and the obtained HPLC detection results are shown in FIG. Figure 1 As shown. Figure 1 It can be seen that there is only one prominent peak, indicating that the product is highly pure.

[0080] The 6'-O-caffeoyl arbutin obtained in Example 1 was detected by a nuclear magnetic resonance instrument, and the obtained nuclear magnetic H spectrum was as follows: Figure 2 The obtained NMR C spectrum is shown in Figure 3 As shown. Figure 2 and Figure 3 It can be seen that the extraction method of the present invention obtains 6'-O-caffeoyl arbutin.

[0081] Example 2

[0082] A method for extracting 6'-O-caffeoyl arbutin comprises the following steps:

[0083] (1) Mixing 30-mesh quezui tea with water and heating and refluxing for 3 times at 110° C., each time for 2 h, to obtain an extract; wherein the mass ratio of quezui tea to water is 1:9;

[0084] (2) the extract obtained in step (1) is concentrated under reduced pressure at 60° C. and −0.08 MPa to obtain an extract concentrate having a relative density of 1.10 g / mL;

[0085] (3) slowly adding an ethanol solution with a mass concentration of 95% to the extract concentrate obtained in step (2) at a stirring rate of 30 rpm / min, wherein the volume ratio of the extract concentrate to the ethanol solution is 1:2.7; after the ethanol solution is completely added, stopping stirring, standing at 5° C. for 12 h for alcohol precipitation, filtering, and washing the obtained suction filtration residue twice with an ethanol solution with a mass concentration of 95%, combining the washing liquid and the suction filtration filtrate to obtain an alcohol precipitate;

[0086] (4) the alcohol precipitate obtained in step (3) was concentrated under reduced pressure at 50° C. and −0.07 MPa to a relative density of 1.15 g / mL, cooled naturally, and then subjected to water precipitation for 12 h at a stirring rate of 20 rpm / min to obtain a water precipitate;

[0087] (5) filtering the water precipitate obtained in step (4), washing the filtered residue with purified water to obtain a solid wet material, dissolving the solid wet material with an ethanol solution with a mass concentration of 60%, wherein the mass ratio of the solid wet material to the ethanol solution is 1:7; filtering, and chromatographically purifying the filtrate to obtain an eluent; wherein the conditions for chromatographic purification are: the chromatographic column filler is a UniPSN 40-300 reverse phase polymer chromatographic column filler, the elution solvent is an ethanol solution with a mass concentration of 35%, the flow rate of the elution solvent is 15 min / BV, the detection wavelength is 326 nm, and the sample load is 5.7 g / L;

[0088] (6) The eluate obtained in step (5) was concentrated under reduced pressure at 50° C. and −0.08 MPa until the volume of the concentrate was 0.06 times the volume of the eluate, and the concentrate was allowed to stand at 5° C. for 12 h for crystallization. The crystals were filtered and washed with water, and then dried in a vacuum drying oven at 90° C. and 10 bar for 4 h to obtain 424.1 g of 6′-O-caffeoyl arbutin.

[0089] Comparative Example 1

[0090] Repeat the test according to the application example 1 in patent CN11035669A. Weigh 100g of the tea sample, and use a 70% methanol aqueous solution at a solid-liquid ratio of g:mL=1:10 for ultrasonic-assisted extraction at 25°C for 30min, filter, extract the residue twice, combine the filtrate, concentrate and freeze-dry to obtain the tea extract.

[0091] Comparative Example 2

[0092] Repeat the test according to Example 1 in patent CN116898778A. 100g of dried quezui tea was heated and refluxed with 60% ethanol-water, the solid-liquid ratio was 1:10g / mL, the extraction temperature was 65℃, the time was 2h, and the extraction times were 2 times; the extract was concentrated under reduced pressure at 55℃ until there was no alcohol taste, cooled at 4℃ for 12h, and the precipitate was fully separated, centrifuged at 3000r / min for 15min, and the precipitate was collected; the precipitate was heated and redissolved with distilled water, loaded on MCI-gelCHP 20P resin for separation and elution, and the separation and elution steps were: water elution 2BV, 20% ethanol-water elution 5BV, 50% ethanol-water elution 5BV; the 50% ethanol-water elution portion was collected, concentrated under reduced pressure at 50℃ to a solid content of 25%, and freeze-dried to obtain.

[0093] Comparative Example 3

[0094] Repeat the test according to the example in patent CN101066985A. 100g of dried quezui tea was extracted by heating and refluxing with 80% ethanol, the solid-liquid ratio was 1:10g / mL, the time was 2h, and the extraction times were 2 times. Filter, combine the filtrate and concentrate, the residual aqueous solution was filtered and defatted, and then further concentrated to remove part of the water, and then placed at room temperature to precipitate, the precipitate was filtered, and the 6′-O-caffeoyl arbutin crude product was obtained by drying. After the above filtrate was concentrated to a small volume, it was chromatographed with macroporous adsorption resin D101, the diameter-to-height ratio of the chromatography column was 1:10, and the weight ratio of the extract to the D101 resin was 1:10. Water (2BV), 20% ethanol (2BV), 30% ethanol (2BV), and 40% ethanol (2BV) were used for gradient elution in sequence to collect the crude product containing 6′-O-caffeoyl arbutin. The crude product was heated and dissolved with an appropriate amount of water, and then placed at room temperature overnight. The precipitated crystals were filtered, washed with water, filtered, and dried to obtain pure 6′-O-caffeoyl arbutin.

[0095] The 6'-O-caffeoyl arbutin obtained in Comparative Examples 1 to 3 was detected by high performance liquid chromatography. The HPLC detection results are shown in FIG. Figures 4 to 6 As shown. Figures 4 to 6 It can be seen that there are multiple prominent peaks in the figure, indicating that there are multiple substances in the product and the purity of the product is low.

[0096] The content of 6'-O-caffeoyl arbutin extracted from Examples 1 to 2 and Comparative Examples 1 to 3 was detected by high performance liquid chromatography:

[0097] The chromatographic conditions are as follows: using octadecylsilane bonded silica gel as filler, using acetonitrile: 0.2% phosphoric acid solution (20:80) as mobile phase for isocratic elution, flow rate of 1.0 ml / min, detection wavelength of 326 nm, theoretical plate number calculated based on 6'-O-caffeoylarbutin peak, should be no less than 5000;

[0098] The determination method is:

[0099] (1) Preparation of reference solution: Accurately weigh an appropriate amount of 6'-O-caffeoyl arbutin reference substance into a volumetric flask, add 75% methanol to dissolve and dilute to prepare a solution containing 0.5 mg per 1 ml;

[0100] (2) Preparation of test solution: Take about 10 mg of the product, weigh it accurately, put it in a stoppered conical flask, add 50 ml of 75% methanol accurately, treat it with ultrasound (power 250 W, frequency 40 kHz) for 15 min, let it cool, weigh it again, make up the loss with 75% methanol, shake it well, filter it, and take the filtrate to obtain the solution;

[0101] (3) Test: Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, determine the peak area, and calculate according to the external standard method.

[0102] The total yield of the extracts obtained in Examples 1-2 and Comparative Examples 1-3 was calculated based on the mass of the product obtained. The purity of the 6'-O-caffeoyl arbutin obtained in Examples 1-2 and Comparative Examples 1-3 was tested using the above method. The results are shown in Table 1.

[0103] Table 1 Total yield of extracts obtained from Examples 1 to 2 and Comparative Examples 1 to 3 and purity of 6'-O-caffeoyl arbutin

[0104] Example Total yield (%) 6'-O-caffeoyl arbutin purity (%) Example 1 66.50 98.56 Example 2 52.01 98.12 Comparative Example 1 96.85 36.54 Comparative Example 2 57.68 74.28 Comparative Example 3 83.30 89.47

[0105] It can be seen from Table 1 that the extraction method provided by the present invention greatly improves the purity of 6'-O-caffeoyl arbutin, and the total yield can also be maintained at a high level.

[0106] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting 6'-O-caffeoyl arbutin, comprising the following steps: (1) mixing a plant of the genus Vaccinium in the family Ericaceae with water and heating the mixture under reflux to obtain an extract; (2) concentrating the extract obtained in step (1) under reduced pressure to obtain an extract concentrate; (3) subjecting the extract concentrate obtained in step (2) to alcohol precipitation to obtain an alcohol precipitate; (4) subjecting the alcohol precipitation liquid obtained in step (3) to water precipitation to obtain a water precipitation liquid; (5) chromatographically purifying the water precipitate obtained in step (4) to obtain an eluent; (6) The eluate obtained in step (5) is concentrated and then crystallized to obtain 6'-O-caffeoyl arbutin.

2. The extraction method according to claim 1, characterized in that The heating reflux temperature in the step (1) is 90 to 110° C., the number of heating reflux is 2 to 5 times, and the time of each heating reflux is 1 to 3 hours.

3. The extraction method according to claim 1, characterized in that The temperature of the reduced pressure concentration in step (2) is 40 to 70° C., the pressure of the reduced pressure concentration is -0.06 to -0.1 MPa, and the endpoint of the reduced pressure concentration is that the relative density of the extracted concentrated solution is 1.10 to 1.25 g / mL.

4. The extraction method according to claim 1, characterized in that In the step (3), the reagent for alcohol precipitation is an ethanol solution, the temperature of alcohol precipitation is 2 to 25° C., and the time of alcohol precipitation is 8 to 24 hours.

5. The extraction method according to claim 4, characterized in that The mass concentration of the ethanol solution is 60-95%, and the volume ratio of the extraction concentrate to the ethanol solution is 1:(2.5-10).

6. The extraction method according to claim 1, characterized in that The water precipitation temperature in step (4) is room temperature, and the water precipitation time is 8 to 24 hours.

7. The extraction method according to claim 1, characterized in that The chromatographic column filler for chromatographic purification in step (5) is a reverse phase C18 chromatographic column filler or a reverse phase polymer chromatographic column filler, the elution solvent for chromatographic purification is an ethanol solution, the detection wavelength for chromatographic purification is 326 nm, the loading amount for chromatographic purification is 3 to 10 g / L, and the chromatographic purification time is from the beginning of the chromatogram corresponding to 6'-O-caffeoyl arbutin to the end of the chromatogram corresponding to 6'-O-caffeoyl arbutin.

8. The extraction method according to claim 7, characterized in that The mass concentration of the ethanol solution is 30-35%, and the flow rate of the ethanol solution is 10-30 min / BV.

9. The extraction method according to claim 1, characterized in that The concentration pressure in step (6) is -0.06 to -0.1 MPa, the concentration temperature is 40 to 70°C, and the concentration endpoint is when the volume of the concentrate is 0.035 to 0.1 times the volume of the eluent.

10. The extraction method according to claim 1, characterized in that The crystallization temperature in step (6) is 2 to 8° C., and the crystallization time is 8 to 18 hours.

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